Quantification of non-reducing end glycan residual compounds

ABSTRACT

Provided herein are methods of diagnosing or monitoring the treatment of abnormal glycan accumulation or a disorder associated with abnormal glycan accumulation.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No. 61/142,291, filed 2 Jan. 2009, U.S. Provisional Application No. 61/164,365, filed 27 Mar. 2009, and U.S. Provisional Application No. 61/238,079, filed 28 Aug. 2009, which applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Many human diseases are caused by or correlated with changes in glycosylation. In order to use these changes as biomarkers of disease, analytical methods are used to quantify the changes. The published methods use antibodies, chromatography and/or mass spectrometry techniques to resolve and quantify the intact or partially intact glycans. These methods are challenging due to the complexity and number of possible glycan structures present in biological samples. In a single disease state there can be thousands of different novel glycan structures that are present; however, each on their own is a weak marker of disease.

SUMMARY OF THE INVENTION

Described herein are populations of glycans that are transformed into populations of biomarkers using glycan degradation enzymes. Further described herein are the use of analytical instruments to characterize the population of biomakers (i.e., non-reducing end glycan residual compounds, such as monosaccharides) in order to provide relevant information about the population of biomarkers, the population of biomarkers and the biological sample that provided the population of biomarkers.

Provided in certain embodiments herein are methods of detecting glycan accumulation and/or abnormal glycan biosynthesis and/or degradation in a biological sample, the method comprising:

-   -   a. transforming a glycan of a biological sample with a glycan         degradation enzyme to liberate a glycan residual compound from         the non-reducing end of the glycan;     -   b. measuring the amount of the glycan residual compound         liberated by the functioning glycan degradation enzyme with an         analytical device.

In some embodiments, a method described herein comprises a method of diagnosing an individual as having a disease or condition associated with abnormal glycan biosynthesis, degradation, or accumulation, the method comprising:

-   -   a. generating a biomarker comprising of one or more non-reducing         end glycan residual compound, wherein the biomarker is generated         by treating a population of glycans, in or isolated from a         biological sample from the individual, with at least one         digesting glycan enzymes, wherein prior to enzyme treatment, the         biomarker is not present in abundance in samples from         individuals with the disease or condition relative to         individuals without the disease or condition, and     -   b. using an analytical instrument to detect the presence of         and/or measure the amount of the biomarker produced and         displaying or recording the presence of or a measure of a         population of the biomarker;

In some embodiments, the presence of and/or measure the amount of the biomarker is utilized to determine the presence, identity, and/or severity of the disease or condition.

Provided in certain embodiments herein is a method of diagnosing an individual as having a disease or condition associated with abnormal glycan biosynthesis, degradation, or accumulation, the method comprising:

-   -   a. transforming a glycan of a biological sample with a glycan         degradation enzyme to liberate a glycan residual compound from         the non-reducing end of the glycan;     -   b. measuring the amount of the glycan residual compound         liberated by the functioning glycan degradation enzyme with an         analytical device; and     -   c. determining whether the amount of liberated glycan residue is         abnormal.

In some embodiments, provided herein is a method of monitoring the treatment of a disorder associated with the abnormal degradation, biosynthesis and/or accumulation of glycans, the method comprising:

-   -   a. following administration of an agent for treating a disorder         associated with the abnormal degradation, biosynthesis and/or         accumulation of glycans to an individual in need thereof, using         an analytical instrument to measure the amount of a population         of a biomarker comprising a non-reducing end glycan residual         compounds present in a transformed biological sample, the         biomarker being generated by treating a population of glycans,         in or isolated from a biological sample from the individual,         with at least one digesting glycan enzyme(s), wherein prior to         enzyme treatment, the biomarker is not present in abundance in         samples from individuals with the disease or condition relative         to individuals without the disease or condition, and     -   b. determining whether or not the amount of the amount of         biomarker has decreased or increased at a slower rate compared         to the amount or rate of increase prior to administration of the         agent for treating a disorder associated with the abnormal         degradation, biosynthesis and/or accumulation of glycans.

In some embodiments, the abnormal glycan accumulation or disorder associated therewith is caused by an abnormally functioning glycan degradation enzyme and wherein the abnormally functioning glycan degradation enzyme and glycan degradation enzyme are of the same type (e.g., the glycan degradation utilized in the transformation process is a functioning glycan degradation enzyme whereas the abnormally functioning enzyme is not, such as due to deletions, insertions, substitutions, or other modifications to the enzyme sequence). In certain embodiments, the abnormally functioning glycan degradation enzyme functions abnormally as a result of being present in an abnormally low amount, functioning improperly, or a combination thereof. In some embodiments, the abnormal glycan accumulation comprises the accumulation of abnormal amounts of glycans. In certain embodiments, the abnormal glycan accumulation comprises the accumulation of abnormal amounts of normal glycans. In some embodiments, the abnormal glycan accumulation comprises the accumulation of abnormal amounts of abnormal glycans.

In certain embodiments, the biomarker is not present in the original biological sample. In some embodiments, the biomarker is not present in the biological sample after isolating a population of glycans therefrom (e.g., prior to transformation of the glycan according to a process described herein).

In certain embodiments, the normally functioning glycan degradation enzyme is a glycosidase, sulfatase, phosphorylase, deacetylase or a combination thereof. In some embodiments, the normally functioning glycan degradation enzyme is a glycosidase selected from an exo-glycosidase and an endo-glycosidase. In certain embodiments, the glycosidase is an exo-glycosidase selected from the group consisting of a galactosidase, and a glucuronidase. In some embodiments, the generated biomarker is a glycan residual compound. In some embodiments, the glycan residual compound is a monosaccharide. In certain embodiments, the glycan residual compound is sulfate, phosphate, acetate, or a combination thereof. In certain embodiments, the glycan residual compound has a molecular weight of less than 2000 g/mol, less than 1500 g/mol, less than 1000 g/mol, less than 500 g/mol, less than 400 g/mol, less than 300 g/mol, less than 260 g/mol, less than 200 g/mol, less than 100 g/mol, or the like (e.g., prior to tagging with any detectable label that may be included in a process described herein).

In some embodiments, any process described herein further comprises purifying a biological sample prior to transforming a glycan thereof. In some embodiments, the process of purifying a biological sample comprises removing monosaccharides therefrom, removing sulfates therefrom, removing phosphates therefrom, removing acetate therefrom, or a combination thereof.

In certain embodiments, transforming a glycan of a biological sample with a normally functioning glycan degradation enzyme comprises transforming a glycan of a biological sample with a plurality of normally functioning glycan degradation enzymes. In some embodiments, the glycan is treated with a plurality of normally functioning glycan degradation enzymes concurrently, sequentially, or a combination thereof.

In specific embodiments, a disorder associated with an abnormal glycan accumulation is any disorder described in Tables 1-4 (e.g., MPS I) and the normally functioning glycan degradation enzyme is any enzyme described in Tables 1-4 (e.g., L-iduronidase).

In some embodiments, determining whether the amount of liberated glycan residue is abnormal comprises labeling the glycan residue with a detectable label and measuring the amount of labeled glycan residue with an analytical instrument. In certain embodiments, the detectable label is a mass label, a radioisotope label, a fluorescent label, a chromophore label, or affinity label. In some embodiments, the amount of liberated glycan is measured using UV-V is spectroscopy, IR spectroscopy, mass spectrometry, or a combination thereof.

Provided in some embodiments herein is a method of monitoring the treatment of a disorder associated with the abnormal degradation, biosynthesis and/or accumulation of glycans, the methods comprising:

-   -   a. following administration of an agent for treating a disorder         associated with the abnormal degradation, biosynthesis and/or         accumulation of glycans to an individual in need thereof, using         an analytical instrument to measure the amount of a population         of a non-reducing end glycan residual compounds present in a         transformed biological sample that has been prepared by:         -   treating a population of glycans, in or isolated from a             biological sample taken from the individual, with at least             one normally functioning glycan degradation enzyme to             liberate non-reducing end glycan residual compound;     -   b. determining whether or not the amount of the amount of         liberated non-reducing end glycan residue has decreased or         increased at a slower rate compared to the amount or rate of         increase prior to administration of the agent for treating a         disorder associated with the abnormal degradation, biosynthesis         and/or accumulation of glycans.

In some embodiments, the disorder associated with the abnormal degradation, biosynthesis and/or accumulation of glycans is a lysosomal storage disease, a cancerous disease, or an infectious disease. In certain embodiments, the normally functioning glycan degradation enzyme is a glycosidase, sulfatase, phosphorylase, deacetylase, or a combination thereof. In some embodiments, the normally functioning glycan degradation enzyme is a glycosidase selected from an exo-glycosidase and an endo-glycosidase. In certain embodiments, the glycan residual compound is a monosaccharide, sulfate, phosphate, acetate, or a combination thereof. In some embodiments, transforming a glycan of a biological sample with a normally functioning glycan degradation enzyme comprises transforming a glycan of a biological sample with a plurality of normally functioning glycan degradation enzymes. In certain embodiments, the glycan is treated with a plurality of normally functioning glycan degradation enzymes concurrently, sequentially, or a combination thereof. In some embodiments, prior to measuring the amount of a population of non-reducing end glycan residual compounds, the non-reducing end glycan residual compounds are labeled with a detectable label. In certain embodiments, the detectable label is a mass label, a radioisotope label, a fluorescent label, a chromophore label, or affinity label. In some embodiments, the amount of liberated glycan is measured using UV-V is spectroscopy, IR spectroscopy, mass spectrometry, or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 illustrates compounds present in a normal biological sample not subject to an enzymatic glycan residual liberation process described herein.

FIG. 2 illustrates compounds present in a normal biological subject to an enzymatic glycan residual liberation process described herein.

FIG. 3 illustrates compounds present in a biological sample of an individual suffering from a disorder associated with abnormal glycan accumulation not subject to an enzymatic glycan residual liberation process described herein.

FIG. 4 illustrates compounds present in a biological sample of an individual suffering from a disorder associated with abnormal glycan accumulation subject to an enzymatic glycan residual liberation process described herein.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Provided herein is a method of detecting abnormal glycan accumulation, e.g., in human disease. In some instances, the process described herein includes a strategy to quantify the changes by measuring the abundance of all glycans with a disease related glycan residual compound on the non-reducing end of glycans from a biological sample (e.g., monosaccharides and/or their modifications such as sulfation, acetylation, phosphorylation, or the like).

Provided in certain embodiments herein are methods of detecting glycan accumulation in a biological sample, the method comprising:

-   -   a. transforming a glycan of a biological sample with a normally         functioning glycan degradation enzyme to liberate a glycan         residual compound from the non-reducing end of the glycan;     -   b. measuring the amount of the glycan residual compound         liberated by the functioning glycan degradation enzyme with an         analytical device.

In certain embodiments, the method is associated with diagnosing an individual with abnormal glycan accumulation, or a disorder associated therewith.

Therefore, in specific embodiments, provided herein is a method of diagnosing an individual as having an abnormal glycan accumulation or a disorder associated with an abnormal glycan accumulation, the method comprising:

-   -   a. transforming a glycan of a biological sample with a normally         functioning glycan degradation enzyme to liberate a glycan         residual compound from the non-reducing end of the glycan;     -   b. measuring the amount of the glycan residual compound         liberated by the functioning glycan degradation enzyme with an         analytical device; and     -   c. determining whether the amount of liberated glycan residue is         abnormal.

In certain instances, methods of detecting abnormal glycan accumulation works based on the observation that altered glycans generated in a disease state are caused by an alteration in the activity of a biosynthetic enzyme (e.g., via increased expression, increased activity, increased substrate, or the like) that leads to the production of thousands of unique structures.

For example, in certain instances, the induction of an alpha 2,3 sialyltransferase leads to the novel expression of thousands of different glycans (potentially from multiple glycan classes) that present a non-reducing terminal alpha 2,3 linked sialic acid. By quantifying a limited set of these novel structures using current methods, only a fraction of the disease related structures are measured. Instead, as provided in certain embodiments herein, if a sample containing glycans (crude or purified for a specific glycan class) is treated with an alpha 2,3 sialidase to liberate the non-reducing end sialic acid, the free sialic acid (non-reducing end glycan residual) can be measured. This signal would represent a larger portion of the thousands of altered glycan structures that are made in the disease state due to the altered expression of the alpha 2,3 sialyltransferase. Furthermore, in certain embodiments, depending on the signal (i.e., measurement) of the sialic acid liberated, a determination is made as to whether or not the accumulation of sialic acid is abnormal and/or whether or not such levels of accumulated sialic acid is associated with a disorder.

Another example of the process includes a method involving a biological sample containing glycans (purified or not) that is treated with an exo-glycosidase (for example a β-galactosidase). In some of such embodiments, enzymatic treatment cleaves non-reducing end monosaccharides within the chosen enzymes specificity (e.g., β-linked galactose residues) and liberates them as free monosaccharide (e.g., galactose). In various embodiments, the free monosaccharide is isolated and quantified by any analytical method (HPLC, MS, GC, etc), and any disease that presents changes in the levels of non-reducing end β-linked galactose residues is detected or diagnosed.

Similar methods are also optionally utilized in methods of monitoring and/or determining the therapeutic of a treatment or treatment regimen, particularly in the treatment of a disorder associated with abnormal glycan accumulation. For example, provided in certain embodiments herein is a method of monitoring the treatment of disorders associated with the abnormal degradation, biosynthesis and/or accumulation of glycans, the methods comprising:

-   -   a. following administration of an agent for treating a disorder         associated with the abnormal degradation, biosynthesis and/or         accumulation of glycans to an individual in need thereof, using         an analytical instrument to measure the amount of a population         of a non-reducing end glycan residue present in a transformed         biological sample that has been prepared by:         -   treating a population of glycans, in or isolated from a             biological sample taken from the individual, with at least             one normally functioning glycan degradation enzyme to             liberate non-reducing end glycan residue;     -   b. determining whether or not the amount of the amount of         liberated non-reducing end glycan residue has decreased or         increased at a slower rate compared to the amount or rate of         increase prior to administration of the agent for treating a         disorder associated with the abnormal degradation, biosynthesis         and/or accumulation of glycans.

In some embodiments, any process described herein comprises:

-   -   a. comparing an amount of a population of one or more glycan         residual compound present in a transformed biological sample to         an amount of a population of one or more glycan residual         compound present in a control biological sample that has been         treated in a manner substantially similar to the transformed         biological sample.

In certain embodiments, a control biological sample utilized in any process described herein was provided from an individual that does not suffer from a disorder being diagnosed. In other embodiments, a control biological sample is taken from an individual suffering from a disorder being diagnosed. In certain embodiments, the result obtained from the control biological sample is stored in a database. In such cases a test sample is optionally compared to a plurality of control data in a database. Moreover in certain embodiments, any diagnostic process described herein is optionally utilized alone or in combination with other diagnostic techniques. Other diagnostic techniques include, by way of non-limiting example, symptom analysis, biopsies, detection of accumulation of other compounds in biological samples, or the like. In some embodiments, control biological samples are optionally taken from the same individual at substantially the same time, simply from a different location (e.g., one inflamed/arthritic synovial joint fluid vs the contralateral non-arthritic synovial joint). In other embodiments, control biological samples are optionally taken from the same individual at different points in time (e.g., before therapy and after therapy if the method being utilized is a method of monitoring a treatment therapy).

Glycan Accumulation:

In various instances, glycan accumulation occurs in a biological sample as a result natural glycan biosynthetic and/or degradation processes. In some instances, abnormal glycan accumulation occurs in a biological sample as a result of a disorder or disease within an individual from which the biological sample is obtained.

In certain embodiments, abnormal glycan accumulation that is observable by methods described herein is associated with the accumulation of glycans in a manner that does not normally occur in individuals who are not in a disease state.

In some embodiments, such accumulation includes the accumulation of abnormal glycans. In certain instances, these abnormal glycans include glycans that are not normally produced in an individual, or a particular biological sample thereof, in the absence of a particular disease state. Therefore, in some embodiments, abnormal glycan accumulation includes the accumulation of glycans, the glycans being abnormal themselves, especially in any significant quantity. In other words, such glycans are abnormal glycans in individuals or particular biological samples thereof when such individuals are in a non-diseased, normal, or wild type state.

In some embodiments, such accumulation includes the abnormal accumulation of glycans. In some instances, these glycans are glycans that normally occur in individuals in a non-diseased state, but at lower or higher levels or are abnormal only due to the location wherein they are produced. Therefore, in some embodiments, abnormal glycan accumulation includes the accumulation of abnormal amounts of glycans or the location thereof, the glycans being normally occurring or abnormal glycans. In other words, the amount of glycan accumulation is abnormal in individuals, or particular biological samples thereof, when such individuals are in a non-diseased, normal, or wild type state.

Biological Sample:

Biological samples suitable for analysis according to the methods and processes described herein include, by way of non-limiting example, blood, serum, urine, hair, saliva, skin, tissue, plasma, cerebrospinal fluid (CSF), amniotic fluid, nipple aspirate, sputum, tears, lung aspirate, semen, feces, synovial fluid, nails, or the like. In specific embodiments, the biological samples suitable for analysis according to the methods and processes described herein include, by way of non-limiting example, urine, serum, plasma, or CSF. In certain embodiments, processes for detecting glycan in a sample comprise providing, from the individual, a test biological sample that comprises glycan. In some embodiments, providing a test biological sample from an individual includes obtaining the sample from the individual or obtaining the sample from another source (e.g., from a technician or institution that obtained the sample from the individual). In some embodiments, the biological sample is obtained from any suitable source, e.g., any tissue or cell (e.g., urine, serum, plasma, or CSF) of an individual. In certain embodiments, the tissue and/or cell from which the glycans are recovered is obtained from liver tissue or cells, brain tissue or cells, kidney tissue or cells, or the like.

In certain embodiments, a biological sample according to any process described herein is taken from any individual. In some embodiments, the individual is an individual suspected of suffering from a disorder associated with abnormal glycan accumulation, biosynthesis, and/or degradation. In certain embodiments, the individual is a newborn or fetus.

In some embodiments, provided herein is a composition comprising isolated glycans, wherein the glycans were isolated from a biological sample, and one or more glycan degradation enzyme. In certain embodiments, the composition further comprises one or more biomarker generated according to any method described herein (e.g., wherein the biomarker is a non-reducing end glycan residual compound). In certain embodiments, provided herein is a biomarker described herein (e.g., a labeled or non-labeled non-reducing end glycan residual compound) and an analytical instrument or chromatographic resin.

Degradation Enzymes:

In certain embodiments, any suitable enzyme is optionally utilized in order to remove a glycan residual compound from the non-reducing end of a glycan. In certain disorders, e.g., as described herein, various types of abnormal glycan accumulation occurs. In certain instances, this type of glycan accumulation is detected and/or measured utilizing any suitable enzyme, e.g., as described herein. For example, Tables 1-4 illustrate various enzymes that are utilized in various embodiments of the processes described herein. Any enzyme with the desired specificity is optionally utilized in any process herein (i.e., to liberate the non-reducing end structures). Enzymes suitable for use in the processes described herein include, by way of non-limiting example, eukaryotic, prokaryotic, native, or recombinant enzymes.

In certain embodiments, a disorder associated with abnormal glycan accumulation includes a disorder associated therewith is caused by an abnormally functioning glycan degradation enzyme. In various embodiments, the abnormally functioning glycan degradation enzyme functions abnormally as a result of being present in an abnormally low amount, functioning improperly, or a combination thereof. For example, an abnormally functioning glycan degradation enzyme functions abnormally as a result of being present in an amount of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% than is present in an individual with normal amounts of the glycan degradation enzyme (e.g., an individual in a non-diseased, normal, or wild type state). In further or alternative embodiments, abnormally functioning glycan degradation enzymes are present in a normal amount, but do not function properly in degrading glycans. For example, such enzymes may be have amino acid substitutions in the sequences thereof that reduce or eliminate the glycan degradative properties of the enzyme.

In some embodiments, wherein abnormal glycan accumulation results, at least partially from, an abnormally functioning glycan degradation enzyme, a normally functioning glycan degradation is optionally utilized, particularly wherein the abnormally functioning glycan degradation enzyme and the normally functioning glycan degradation enzyme are of the same type.

Normally functioning glycan degradation enzymes that are used in various embodiments described herein include, by way of non-limiting example, glycosidases, sulfatases, phosphorylases, deacetylases, sialidases, or combinations thereof. In more specific embodiments, a normally functioning glycan degradation enzyme is a glycosidase, e.g., an exo-glycosidase or an endo-glycosidase. In more specific embodiments, the glycosidase is an exo-glycosidase, e.g., galactosidase, and a glucuronidase. In some embodiments, such enzymes serve to remove various glycan residual compounds, such as, monosaccharides, sulfate, phosphate, acetate, sialic acid, or combinations thereof, which are detected and/or measured in methods described herein.

In certain embodiments, one or normally functioning glycan degradation enzyme is optionally utilized to liberate a targeted glycan residual compound. Multiple enzyme treatments of glycans within a biological sample are useful in various embodiments, e.g., wherein a particular enzyme is unable to liberate a targeted residual glycan compound without first modifying the non-reducing end of the glycan. For example, a first enzyme is optionally utilized to remove a sulfate so that a second enzyme can be utilized to remove a monosaccharide. In various embodiments, the glycans are treated with a plurality of normally functioning glycan degradation enzymes concurrently, sequentially, or a combination thereof.

Various enzymes that are used in various embodiments of the methods described herein include, by way of non-limiting example, a glycosidase. Non-limiting examples of glycosidase that are optionally utilized in the methods described herein include, by way of non-limiting example, enzymes categorized as 3.2.1.X by BRENDA (the comprehensive Enzyme Information System) including 3.2.1.1 alpha-amylase, 3.2.1.B1 extracellular agarase, 3.2.1.2 beta-amylase, 3.2.1.3 glucan 1,4-alpha-glucosidase, 3.2.1.4 cellulase, 3.2.1.5 licheninase, 3.2.1.6 endo-1,3(4)-beta-glucanase, 3.2.1.7 inulinase, 3.2.1.8 endo-1,4-beta-xylanase, 3.2.1.9 amylopectin-1,6-glucosidase, 3.2.1.10 oligo-1,6-glucosidase, 3.2.1.11 dextranase, 3.2.1.12 cycloheptaglucanase, 3.2.1.13 cyclohexaglucanase, 3.2.1.14 chitinase, 3.2.1.15 polygalacturonase, 3.2.1.16 alginase, 3.2.1.17 lysozyme, 3.2.1.18 exo-alpha-sialidase, 3.2.1.19 heparinase, 3.2.1.20 alpha-glucosidase, 3.2.1.21 beta-glucosidase, 3.2.1.22 alpha-galactosidase, 3.2.1.23 beta-galactosidase, 3.2.1.24 alpha-mannosidase, 3.2.1.25 beta-mannosidase, 3.2.1.26 beta-fructofuranosidase, 3.2.1.27 alpha-1,3-glucosidase, 3.2.1.28 alpha,alpha-trehalase, 3.2.1.29 chitobiase, 3.2.1.30 beta-D-acetylglucosaminidase, 3.2.1.31 beta-glucuronidase, 3.2.1.32 xylan endo-1,3-beta-xylosidase, 3.2.1.33 amylo-alpha-1,6-glucosidase, 3.2.1.34 chondroitinase, 3.2.1.35 hyaluronoglucosaminidase, 3.2.1.36 hyaluronoglucuronidase, 3.2.1.37 xylan 1,4-beta-xylosidase, 3.2.1.38 beta-D-fucosidase, 3.2.1.39 glucan endo-1,3-beta-D-glucosidase, 3.2.1.40 alpha-L-rhamnosidase, 3.2.1.41 pullulanase, 3.2.1.42 GDP-glucosidase, 3.2.1.43 beta-L-rhamnosidase, 3.2.1.44 fucoidanase, 3.2.1.45 glucosylceramidase, 3.2.1.46 galactosylceramidase, 3.2.1.47 galactosylgalactosylglucosylceramidase, 3.2.1.48 sucrose alpha-glucosidase, 3.2.1.49 alpha-N-acetylgalactosaminidase, 3.2.1.50 alpha-N-acetylglucosaminidase, 3.2.1.51 alpha-L-fucosidase, 3.2.1.52 beta-N-acetylhexosaminidase, 3.2.1.53 beta-N-acetylgalactosaminidase, 3.2.1.54 cyclomaltodextrinase, 3.2.1.55 alpha-N-arabinofuranosidase, 3.2.1.56 glucuronosyl-disulfoglucosamine glucuronidase, 3.2.1.57 isopullulanase, 3.2.1.58 glucan 1,3-beta-glucosidase, 3.2.1.59 glucan endo-1,3-alpha-glucosidase, 3.2.1.60 glucan 1,4-alpha-maltotetraohydrolase, 3.2.1.61 mycodextranase, 3.2.1.62 glycosylceramidase, 3.2.1.63 1,2-alpha-L-fucosidase, 3.2.1.64 2,6-beta-fructan 6-levanbiohydrolase, 3.2.1.65 levanase, 3.2.1.66 quercitrinase, 3.2.1.67 galacturan 1,4-alpha-galacturonidase, 3.2.1.68 isoamylase, 3.2.1.69 amylopectin 6-glucanohydrolase, 3.2.1.70 glucan 1,6-alpha-glucosidase, 3.2.1.71 glucan endo-1,2-beta-glucosidase, 3.2.1.72 xylan 1,3-beta-xylosidase, 3.2.1.73 licheninase, 3.2.1.74 glucan 1,4-beta-glucosidase, 3.2.1.75 glucan endo-1,6-beta-glucosidase, 3.2.1.76 L-iduronidase, 3.2.1.77 mannan 1,2-(1,3)-alpha-mannosidase, 3.2.1.78 mannan endo-1,4-beta-mannosidase, 3.2.1.79 alpha-L-arabinofuranoside hydrolase, 3.2.1.80 fructan beta-fructosidase, 3.2.1.81 beta-agarase, 3.2.1.82 exo-poly-alpha-galacturonosidase, 3.2.1.83 kappa-carrageenase, 3.2.1.84 glucan 1,3-alpha-glucosidase, 3.2.1.85 6-phospho-beta-galactosidase, 3.2.1.86 6-phospho-beta-glucosidase, 3.2.1.87 capsular-polysaccharide endo-1,3-alpha-galactosidase, 3.2.1.88 beta-L-arabinosidase, 3.2.1.89 arabinogalactan endo-1,4-beta-galactosidase, 3.2.1.90 arabinogalactan endo-1,3-beta-galactosidase, 3.2.1.91 cellulose 1,4-beta-cellobiosidase, 3.2.1.92 peptidoglycan beta-N-acetylmuramidase, 3.2.1.93 alpha,alpha-phosphotrehalase, 3.2.1.94 glucan 1,6-alpha-isomaltosidase, 3.2.1.95 dextran 1,6-alpha-isomaltotriosidase, 3.2.1.96 mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase, 3.2.1.97 glycopeptide alpha-N-acetylgalactosaminidase, 3.2.1.98 glucan 1,4-alpha-maltohexaosidase, 3.2.1.99 arabinan endo-1,5-alpha-L-arabinosidase, 3.2.1.100 mannan 1,4-mannobiosidase, 3.2.1.101 mannan endo-1,6-alpha-mannosidase, 3.2.1.102 blood-group-substance endo-1,4-beta-galactosidase, 3.2.1.103 keratan-sulfate endo-1,4-beta-galactosidase, 3.2.1.104 steryl-beta-glucosidase, 3.2.1.105 3alpha(S)-strictosidine beta-glucosidase, 3.2.1.106 mannosyl-oligosaccharide glucosidase, 3.2.1.107 protein-glucosylgalactosylhydroxylysine glucosidase, 3.2.1.108 lactase, 3.2.1.109 endogalactosaminidase, 3.2.1.110 mucinaminylserine mucinaminidase, 3.2.1.111 1,3-alpha-L-fucosidase, 3.2.1.112 2-deoxyglucosidase, 3.2.1.113 mannosyl-oligosaccharide 1,2-alpha-mannosidase, 3.2.1.114 mannosyl-oligosaccharide 1,3-1,6-alpha-mannosidase, 3.2.1.115 branched-dextran exo-1,2-alpha-glucosidase, 3.2.1.116 glucan 1,4-alpha-maltotriohydrolase, 3.2.1.117 amygdalin beta-glucosidase, 3.2.1.118 prunasin beta-glucosidase, 3.2.1.119 vicianin beta-glucosidase, 3.2.1.120 oligoxyloglucan beta-glycosidase, 3.2.1.121 polymannuronate hydrolase, 3.2.1.122 maltose-6′-phosphate glucosidase, 3.2.1.123 endoglycosylceramidase, 3.2.1.124 3-deoxy-2-octulosonidase, 3.2.1.125 raucaffricine beta-glucosidase, 3.2.1.126 coniferin beta-glucosidase, 3.2.1.127 1,6-alpha-L-fucosidase, 3.2.1.128 glycyrrhizinate beta-glucuronidase, 3.2.1.129 endo-alpha-sialidase, 3.2.1.130 glycoprotein endo-alpha-1,2-mannosidase, 3.2.1.131 xylan alpha-1,2-glucuronosidase, 3.2.1.132 chitosanase, 3.2.1.133 glucan 1,4-alpha-maltohydrolase, 3.2.1.134 difructose-anhydride synthase, 3.2.1.135 neopullulanase, 3.2.1.136 glucuronoarabinoxylan endo-1,4-beta-xylanase, 3.2.1.137 mannan exo-1,2-1,6-alpha-mannosidase, 3.2.1.138 anhydrosialidase, 3.2.1.139 alpha-glucuronidase, 3.2.1.140 lacto-N-biosidase, 3.2.1.141 4-alpha-D-{(1->4)-alpha-D-glucano}trehalose trehalohydrolase, 3.2.1.142 limit dextrinase, 3.2.1.143 poly(ADP-ribose) glycohydrolase, 3.2.1.144 3-deoxyoctulosonase, 3.2.1.145 galactan 1,3-beta-galactosidase, 3.2.1.146 beta-galactofuranosidase, 3.2.1.147 thioglucosidase, 3.2.1.148 ribosylhomocysteinase, 3.2.1.149 beta-primeverosidase, 3.2.1.150 oligoxyloglucan reducing-end-specific cellobiohydrolase, 3.2.1.151 xyloglucan-specific endo-beta-1,4-glucanase, 3.2.1.152 mannosylglycoprotein endo-beta-mannosidase, 3.2.1.153 fructan beta-(2,1)-fructosidase, 3.2.1.154 fructan beta-(2,6)-fructosidase, 3.2.1.155 xyloglucan-specific exo-beta-1,4-glucanase, 3.2.1.156 oligosaccharide reducing-end xylanase, 3.2.1.157 iota-carrageenase 3.2.1.158 alpha-agarase, 3.2.1.159 alpha-neoagaro-oligosaccharide hydrolase, 3.2.1.160 xyloglucan-specific exo-beta-1,4-glucanase, 3.2.1.161 beta-apiosyl-beta-glucosidase, 3.2.1.162 lambda-carrageenase, 3.2.1.163 1,6-alpha-D-mannosidase, 3.2.1.164 galactan endo-1,6-beta-galactosidase, 3.2.1.165 exo-1,4-beta-D-glucosaminidase, or a combination thereof.

Other enzymes that are used in various embodiments of the methods described herein include, by way of non-limiting example, a sulfatase including, e.g., enzymes categorized as 3.1.6.X by BRENDA (the comprehensive Enzyme Information System) including 3.1.6.1 arylsulfatase, 3.1.6.2 steryl-sulfatase, 3.1.6.3 glycosulfatase, 3.1.6.4 N-acetylgalactosamine-6-sulfatase, 3.1.6.5 sinigrin sulfohydrolase; myrosulfatase, 3.1.6.6 choline-sulfatase, 3.1.6.7 cellulose-polysulfatase, 3.1.6.8 cerebroside-sulfatase, 3.1.6.9 chondro-4-sulfatase, 3.1.6.10 chondro-6-sulfatase, 3.1.6.11 disulfoglucosamine-6-sulfatase, 3.1.6.12 N-acetylgalactosamine-4-sulfatase, 3.1.6.13 iduronate-2-sulfatase, 3.1.6.14 N-acetylglucosamine-6-sulfatase, 3.1.6.15 N-sulfoglucosamine-3-sulfatase, 3.1.6.16 monomethyl-sulfatase, 3.1.6.17 D-lactate-2-sulfatase, 3.1.6.18 glucuronate-2-sulfatase, 3.10.1.1 N-sulfoglucosamine sulfohydrolase, or combinations thereof.

Certain enzymes that are used in various embodiments of the methods described herein include, by way of non-limiting example, a deacetylase, e.g., an exo-deacetylase, including, by way of non-limiting example, the alpha-glucosaminide N-acetyltransferase (2.3.1.78) or similar enzymes.

Certain enzymes that are used in various embodiments of the methods described herein include, by way of non-limiting example, a carbohydrate phosphatase including, e.g., 3.1.3.1 alkaline phosphatase, 3.1.3.2 acid phosphatase, 3.1.3.B2 diacylglycerol pyrophosphate phosphatase, 3.1.3.3 phosphoserine phosphatase, 3.1.3.4 phosphatidate phosphatase, 3.1.3.5 5′-nucleotidase, 3.1.3.6 3′-nucleotidase, 3.1.3.7 3′(2′),5′-bisphosphate nucleotidase, 3.1.3.8 3-phytase, 3.1.3.9 glucose-6-phosphatase, 3.1.3.10 glucose-1-phosphatase, 3.1.3.11 fructose-bisphosphatase, 3.1.3.12 trehalose-phosphatase, 3.1.3.13 bisphosphoglycerate phosphatase, 3.1.3.14 methylphosphothioglycerate phosphatase, 3.1.3.15 histidinol-phosphatase, 3.1.3.16 phosphoprotein phosphatase, 3.1.3.17 [phosphorylase] phosphatase, 3.1.3.18 phosphoglycolate phosphatase, 3.1.3.19 glycerol-2-phosphatase, 3.1.3.20 phosphoglycerate phosphatase, 3.1.3.21 glycerol-1-phosphatase, 3.1.3.22 mannitol-1-phosphatase, 3.1.3.23 sugar-phosphatase, 3.1.3.24 sucrose-phosphate phosphatase, 3.1.3.25 inositol-phosphate phosphatase, 3.1.3.26 4-phytase, 3.1.3.27 phosphatidylglycerophosphatase, 3.1.3.28 ADP-phosphoglycerate phosphatase, 3.1.3.29 N-acylneuraminate-9-phosphatase, 3.1.3.30 3′-phosphoadenylylsulfate 3′-phosphatase, 3.1.3.31 nucleotidase, 3.1.3.32 polynucleotide 3′-phosphatase, 3.1.3.33 polynucleotide 5′-phosphatase, 3.1.3.34 deoxynucleotide 3′-phosphatase, 3.1.3.35 thymidylate 5′-phosphatase, 3.1.3.36 phosphoinositide 5-phosphatase, 3.1.3.37 sedoheptulose-bisphosphatase, 3.1.3.38 3-phosphoglycerate phosphatase, 3.1.3.39 streptomycin-6-phosphatase, 3.1.3.40 guanidinodeoxy-scyllo-inositol-4-phosphatase, 3.1.3.41 4-nitrophenylphosphatase, 3.1.3.42 [glycogen-synthase-D] phosphatase, 3.1.3.43 [pyruvate dehydrogenase (acetyl-transferring)]-phosphatase, 3.1.3.44 [acetyl-CoA carboxylase]-phosphatase, 3.1.3.45 3-deoxy-manno-octulosonate-8-phosphatase, 3.1.3.46 fructose-2,6-bisphosphate 2-phosphatase, 3.1.3.47 [hydroxymethylglutaryl-CoA reductase (NADPH)]-phosphatase, 3.1.3.48 protein-tyrosine-phosphatase, 3.1.3.49 [pyruvate kinase]-phosphatase, 3.1.3.50 sorbitol-6-phosphatase, 3.1.3.51 dolichyl-phosphatase, 3.1.3.52 [3-methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring)]-phosphatase, 3.1.3.53 [myosin-light-chain] phosphatase, 3.1.3.54 fructose-2,6-bisphosphate 6-phosphatase, 3.1.3.55 caldesmon-phosphatase, 3.1.3.56 inositol-polyphosphate 5-phosphatase, 3.1.3.57 inositol-1,4-bisphosphate 1-phosphatase, 3.1.3.58 sugar-terminal-phosphatase, 3.1.3.59 alkylacetylglycerophosphatase, 3.1.3.60 phosphoenolpyruvate phosphatase, 3.1.3.61 inositol-1,4,5-trisphosphate 1-phosphatase, 3.1.3.62 multiple inositol-polyphosphate phosphatase, 3.1.3.632-carboxy-D-arabinitol-1-phosphatase, 3.1.3.64 phosphatidylinositol-3-phosphatase, 3.1.3.65 inositol-1,3-bisphosphate 3-phosphatase, 3.1.3.66 phosphatidylinositol-3,4-bisphosphate 4-phosphatase, 3.1.3.67 phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase, 3.1.3.68 2-deoxyglucose-6-phosphatase, 3.1.3.69 glucosylglycerol 3-phosphatase, 3.1.3.70 mannosyl-3-phosphoglycerate phosphatase, 3.1.3.71 2-phosphosulfolactate phosphatase, 3.1.3.72 5-phytase, 3.1.3.73 alpha-ribazole phosphatase, 3.1.3.74 pyridoxal phosphatase, 3.1.3.75 phosphoethanolamine/phosphocholine phosphatase, 3.1.3.76 lipid-phosphate phosphatase, 3.1.3.77 acireductone synthase, 3.1.3.78 phosphatidylinositol-4,5-bisphosphate 4-phosphatase, or 3.1.3.79 mannosylfructose-phosphate phosphatase, or a combination thereof.

In some embodiments, processes described herein include incubation and digestion with a first enzyme to clear a specific non-reducing end structure, incubation and digestion with a second enzyme. In certain embodiments, this multi-enzyme approach is useful in order to reduce the background. For example, in MPS II treating the sample with an iduronidase and/or glucuronidase to clear all non-sulfated non-reducing end uronic acids (this enzyme will not cleave sulfated iduronic acids) before 2-O sulfatase treatment. This approach will clear all non-sulfated non-reducing end uronic acids so that upon desulfation with the 2-O sulfatase the newly releasable uronic acids will be those that were previously sulfated (and therefore resistant to the action of the iduronidase and/or glucuronidase).

Glycan Residual Compounds:

Glycan residual compounds detected, measured, analyzed, and/or otherwise characterized according to any process described herein include any suitable glycan residue that is liberated from the non-reducing end of a glycan (e.g., a glycan obtained from a biological sample of an individual). In specific instances, glycan residual compounds including, e.g., oligosaccharides, monosaccharides, sulfate, phosphate, sialic acid, acetate, or the like.

Specific glycan residual compounds useful in any process herein are described in Tables 1-4.

In some embodiments, the generated biomarker is a glycan residual compound. In some embodiments, the glycan residual compound is a monosaccharide. In certain embodiments, the glycan residual compound is sulfate, phosphate, acetate, or a combination thereof. In certain embodiments, the glycan residual compound has a molecular weight of less than 2000 g/mol, less than 1500 g/mol, less than 1000 g/mol, less than 500 g/mol, less than 400 g/mol, less than 300 g/mol, less than 260 g/mol, less than 200 g/mol, less than 100 g/mol, or the like (e.g., prior to tagging with any detectable label that may be included in a process described herein).

Disorders:

In certain embodiments, a disorder associated with abnormal glycan accumulation includes a disorder associated therewith is caused by an abnormally functioning glycan degradation enzyme. In various embodiments, the abnormally functioning glycan degradation enzyme functions abnormally as a result of being present in an abnormally low amount, functioning improperly, or a combination thereof. For example, an abnormally functioning glycan degradation enzyme functions abnormally as a result of being present in an amount of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% than is present in an individual with normal amounts of the glycan degradation enzyme (e.g., an individual in a non-diseased, normal, or wild type state). In further or alternative embodiments, abnormally functioning glycan degradation enzymes are present in a normal amount, but do not function properly in degrading glycans. For example, such enzymes may be have amino acid substitutions in the sequences thereof that reduce or eliminate the glycan degradative properties of the enzyme.

MPS I is a human genetic disease caused by a deficiency in the lysosomal enzyme L-iduronidase. This enzyme is required in the lysosome to degrade glycans that contain iduronic acid. Due to this enzymatic deficiency, glycans with an iduronic acid on the non-reducing end accumulate to high levels (including heparan sulfate and dermatan sulfate). In certain embodiments, using the method described herein, MPS I is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed into a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with water or buffer) to remove free monosaccharides, then treated with an iduronidase (e.g., to liberate a glycan residual compound iduronic acid). In certain embodiments, after incubation, the liberated iduronic acid is isolated, e.g., by washing the free monosaccharide through the defined MW cut off membrane (or other methods). In some of such embodiments, the monosaccharide would be in the flow through. The isolated monosaccharide solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for iduronic acid content by any suitable analytical technique (e.g., HPLC, MS, GC, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS I disease, measure disease severity, or to measure response to therapy.

MPS II is a human genetic disease caused by a deficiency in the lysosomal enzyme 2-sulfatase. This enzyme is required in the lysosome to degrade glycans that contain 2-O sulfated uronic acids. Due to this enzymatic deficiency, glycans with a 2-sulfated uronic acid on the non-reducing end accumulate to high levels (including heparan sulfate and dermatan sulfate). In certain embodiments, using the method described herein, MPS II is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer to remove free sulfate), and treated with a 2-sulfatase (e.g., to liberate a glycan residual compound sulfate). In some embodiments, after incubation, the liberated sulfate is optionally isolated by washing the free monosaccharide (e.g., through a defined MW cut off membrane or by any other suitable method). In some of such embodiments, the free sulfate is in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for sulfate content by any suitable analytical technique (e.g., HPLC, MS, GC, pH detection, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS II disease, measure disease severity, or to measure response to therapy. In other exemplary embodiments, following treatment with a 2-sulfatase, the resulting 2-O desulfated non-reducing end uronic acid residues is optionally liberated with an iduronidase or glucuronidase. In some of such embodiments, the resulting liberated monosaccharide is optionally isolated, e.g., by washing free monosaccharide (e.g., through the defined MW cut off membrane or any other suitable method). In some of such embodiments, free iduronic or glucuronic acid is in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for monosaccharide content by any suitable analytical technique (e.g., HPLC, MS, GC, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS II disease, measure disease severity, or to measure response to therapy.

MPS IIIA is a human genetic disease caused by a deficiency in the lysosomal enzyme N-sulfatase. This enzyme is required in the lysosome to degrade glycans that contain N-sulfated glucosamine residues. Due to this enzymatic deficiency, glycans with N-sulfated glucosamine residues on the non-reducing end accumulate to high levels (including heparan sulfate). In certain embodiments, using the method described herein, MPS IIIA is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer) to remove free sulfate, and treated with an N-sulfatase. In certain embodiments, after incubation, the liberated sulfate is optionally isolated, e.g., by washing the free monosaccharide (such as through a defined MW cut off membrane or any other suitable method). In some of such embodiments, free sulfate for detection and/or quantitation in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for sulfate content by any suitable analytical technique (e.g., HPLC, MS, GC, pH detection, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS IIIA disease, measure disease severity, or to measure response to therapy. In further or alternative embodiments, following treatment with an N-sulfatase, the resulting N-desulfated non-reducing end glucosamine residues is optionally liberated with a hexosaminidase. In some of such embodiments, liberated monosaccharide is optionally isolated (e.g., by washing the free monosaccharide, such as through the defined MW cut off membrane or any other suitable method). In some of such embodiments, free glucosamine for detection and/or quantitation is present in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for monosaccharide content by any suitable analytical technique (e.g., HPLC, MS, GC, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS IIIA disease, measure disease severity, or to measure response to therapy.

As discussed above, in certain embodiments, using the method described herein, MPS IIIA is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer) to remove free monosaccharide, and treated with an N-sulfo glucosaminidase such as a heparin lyase. In some embodiments, liberated sulfated monosaccharide is optionally isolated, e.g., by washing the free monosaccharide (such as through the defined MW cut off membrane or by any other suitable method). In some of such embodiments, free N-sulfated glucosamine for detection and/or quantitation is present in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for monosaccharide content by any suitable analytical technique (e.g., HPLC, MS, GC, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS IIIA disease, measure disease severity, or to measure response to therapy.

As discussed above, in certain embodiments, using the method described herein, MPS IIIA is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer) to remove free monosaccharide, and treated with an N-sulfatase. In certain embodiments, the resulting glycan is subsequently treated such that the N-desulfated non-reducing end glucosamine residues is acetylated (e.g., with an N-acetyl transferase) and subsequently liberated with a hexosaminidase. In some of such embodiments, the resulting liberated monosaccharide is optionally isolated, e.g., by washing the free monosaccharide (e.g., through a defined MW cut off membrane or any other suitable methods). In some of such embodiments, free N-acetyl glucosamine for detection and/or quantitation is present in the flow through. In certain embodiments, the resulting isolated composition is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for monosaccharide content by any suitable analytical technique (e.g., HPLC, MS, GC, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS IIIA disease, measure disease severity, or to measure response to therapy.

MPS IIIB is a human genetic disease caused by a deficiency in the enzyme N-acetyl glucosaminidase. This enzyme is required in the lysosome to degrade glycans that contain N-acetyl glucosamine residues. Due to this enzymatic deficiency, glycans with a N-acetyl glucosamine residue on the non-reducing end accumulate to high levels (including heparan sulfate). In certain embodiments, using the method described herein, MPS IIIB is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer to remove free N-acetyl glucosamine), and treated with a-acetyl glucosaminidase or a heparin lyase (e.g., to liberate a glycan residual compound N-acetyl glucosamine). In some embodiments, after incubation, the liberated N-acetyl glucosamine is optionally isolated by washing the free monosaccharide (e.g., through a defined MW cut off membrane or by any other suitable method). In some of such embodiments, the free monosaccharide is in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for monosaccharide content by any suitable analytical technique (e.g., HPLC, MS, GC, pH detection, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS IIIB disease, measure disease severity, or to measure response to therapy.

As discussed above, in certain embodiments, using the method described herein, MPS IIIA is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer) to remove free acetate, and treated with a deacetylase. The liberated acetate is optionally isolated, e.g., by washing the free acetate (such as through the defined MW cut off membrane or any other suitable method). In some of such embodiments, the free acetate for detection and/or quantitation is present the flow through. In some embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for acetate content by any suitable analytical technique (e.g., HPLC, MS, GC, pH detection, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS IIIB disease, measure disease severity, or to measure response to therapy.

MPS IIIC is a human genetic disease caused by a deficiency in the enzyme N-acetyltransferase. This enzyme is required in the lysosome to degrade glycans that contain glucosamine residues. Due to this enzymatic deficiency, glycans with a glucosamine residue on the non-reducing end accumulate to high levels (including heparan sulfate). In certain embodiments, using the method described herein, MPS IIIC is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer to remove free glucosamine), and treated with a hexosaminidase or heparin lyase (e.g., to liberate a glycan residual compound glucosamine). In some embodiments, after incubation, the liberated glucosamine is optionally isolated by washing the free glucosamine (e.g., through a defined MW cut off membrane or by any other suitable method). In some of such embodiments, the free glucosamine for detection and/or quantitation is present in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for monosaccharide content by any suitable analytical technique (e.g., HPLC, MS, GC, pH detection, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS IIIC disease, measure disease severity, or to measure response to therapy.

As discussed above, in certain embodiments, using the method described herein, MPS IIIC is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer to remove free glucosamine and/or N-acetyl glucosamine), and treated with a glucosamine N-acetyltransferase followed by a hexosaminidase (e.g., to liberate a glycan residual compound N-acetyl glucosamine). In some embodiments, after incubation, the liberated N-acetyl glucosamine is optionally isolated by washing the free N-acetyl glucosamine (e.g., through a defined MW cut off membrane or by any other suitable method). In some of such embodiments, the free N-acetyl glucosamine for detection and/or quantitation is present in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for monosaccharide content by any suitable analytical technique (e.g., HPLC, MS, GC, pH detection, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS IIIC disease, measure disease severity, or to measure response to therapy.

MPS IIID is a human genetic disease caused by a deficiency in the enzyme glucosamine 6-O sulfatase. This enzyme is required in the lysosome to degrade glycans that contain 6-O-sulfated glucosamine residues. Due to this enzymatic deficiency, glycans with a 6-O-sulfated N-acetyl glucosamine residue on the non-reducing end accumulate to high levels (including heparan sulfate). In certain embodiments, using the method described herein, MPS IIIC is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer to remove free sulfate), and treated with a 6-O-sulfatase (e.g., to liberate a glycan residual compound sulfate). In some embodiments, after incubation, the liberated sulfate is optionally isolated by washing the free sulfate (e.g., through a defined MW cut off membrane or by any other suitable method). In some of such embodiments, the free sulfate for detection and/or quantitation is present in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for sulfate content by any suitable analytical technique (e.g., HPLC, MS, GC, pH detection, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS IIID disease, measure disease severity, or to measure response to therapy.

As discussed above, in certain embodiments, using the method described herein, MPS IIID is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer to remove free sulfate and/or N-acetyl glucosamine), and treated with a 6-O-sulfatase and a hexosaminidase (e.g., to liberate a glycan residual compound N-acetyl glucosamine). In some embodiments, after incubation, the liberated N-acetyl glucosamine is optionally isolated by washing the free N-acetyl glucosamine (e.g., through a defined MW cut off membrane or by any other suitable method). In some of such embodiments, the free monosaccharide for detection and/or quantitation is present in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for monosaccharide content by any suitable analytical technique (e.g., HPLC, MS, GC, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS IIID disease, measure disease severity, or to measure response to therapy.

As discussed above, in certain embodiments, using the method described herein, MPS IIID is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer to remove free sulfate and/or N-acetyl glucosamine 6-O sulfate), and treated with a hexosaminidase or heparin lyase (e.g., to liberate a glycan residual compound N-acetyl glucosamine 6-O sulfate). In some embodiments, after incubation, the liberated N-acetyl glucosamine 6-O sulfate is optionally isolated by washing the free N-acetyl glucosamine 6-O sulfate (e.g., through a defined MW cut off membrane or by any other suitable method). In some of such embodiments, the free monosaccharide for detection and/or quantitation is present in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for monosaccharide content by any suitable analytical technique (e.g., HPLC, MS, GC, pH detection, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS IIID disease, measure disease severity, or to measure response to therapy.

MPS IVA is a human genetic disease caused by a deficiency in the enzyme lysosomal enzyme galactose/N-acetyl galactosamine 6-O sulfatase. This enzyme is required in the lysosome to degrade glycans that contain 6-O-sulfated galactose and 6-O sulfated N-acetyl galactosamine residues. Due to this enzymatic deficiency, glycans with 6-O-sulfated galactose and 6-O sulfated N-acetyl galactosamine residues on the non-reducing end accumulate to high levels (including chondroitin and keratan sulfate). In certain embodiments, using the method described herein, MPS IVA is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer to remove free monosaccharide), and treated with a galactose 6-O-sulfatase and/or an N-acetyl galactosamine 6-O sulfatase and a galactosidase and/or hexosaminidase (e.g., to liberate a glycan residual compound Gal and/or GalNAc). In some embodiments, after incubation, the liberated monosaccharide is optionally isolated by washing the free monosaccharide (e.g., through a defined MW cut off membrane or by any other suitable method). In some of such embodiments, the free monosaccharide for detection and/or quantitation is present in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for monosaccharide content by any suitable analytical technique (e.g., HPLC, MS, GC, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS IVA disease, measure disease severity, or to measure response to therapy.

As discussed above, in certain embodiments, using the method described herein, MPS IVA is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer to remove free sulfate), and treated with a 6-O-sulfatase capable of desulfating 6-O-sulfated galactose and/or 6-O sulfated N-acetyl galactosamine residues (e.g., to liberate a glycan residual compound sulfate). In some embodiments, after incubation, the liberated sulfate is optionally isolated by washing the free sulfate (e.g., through a defined MW cut off membrane or by any other suitable method). In some of such embodiments, the free sulfate for detection and/or quantitation is present in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for sulfate content by any suitable analytical technique (e.g., HPLC, MS, GC, pH detection, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS IVA disease, measure disease severity, or to measure response to therapy.

MPS IVB is a human genetic disease caused by a deficiency in the enzyme lysosomal β-galactosidase. This enzyme is required in the lysosome to degrade glycans that contain galactose residues. Due to this enzymatic deficiency, glycans with β-galactose residues on the non-reducing end accumulate to high levels (including keratan sulfate and other glycans). In certain embodiments, using the method described herein, MPS IVB is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer to remove free monosaccharide), and treated with a galactosidase (e.g., to liberate a glycan residual compound Gal). In some embodiments, after incubation, the liberated monosaccharide is optionally isolated by washing the free monosaccharide (e.g., through a defined MW cut off membrane or by any other suitable method). In some of such embodiments, the free monosaccharide for detection and/or quantitation is present in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for monosaccharide content by any suitable analytical technique (e.g., HPLC, MS, GC, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS IVB disease, measure disease severity, or to measure response to therapy.

MPS VI is a human genetic disease caused by a deficiency in the enzyme 4-O sulfatase that desulfates N-acetyl galactosamine. This enzyme is required in the lysosome to degrade glycans that contain 4-O-sulfated N-acetyl galactosamine residues. Due to this enzymatic deficiency, glycans with 4-O-sulfated N-acetyl galactosamine residues on the non-reducing end accumulate to high levels (including chondroitin sulfate). In certain embodiments, using the method described herein, MPS VI is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer to remove free sulfate), and treated with a 4-O-sulfatase that can desulfate 4-O-sulfated N-acetyl galactosamine residues (e.g., to liberate a glycan residual compound sulfate). In some embodiments, after incubation, the liberated sulfate is optionally isolated by washing the free sulfate (e.g., through a defined MW cut off membrane or by any other suitable method). In some of such embodiments, the free sulfate for detection and/or quantitation is present in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for sulfate content by any suitable analytical technique (e.g., HPLC, MS, GC, pH detection, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS VI disease, measure disease severity, or to measure response to therapy.

As discussed above, in certain embodiments, using the method described herein, MPS VI is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer to remove free N-acetyl galactosamine), and treated with a 4-O-sulfatase that is capable of desulfating 4-O-sulfated N-acetyl galactosamine residues then treated with a hexosaminidase (e.g., to liberate a glycan residual compound N-acetyl galactosamine). In some embodiments, after incubation, the liberated N-acetyl galactosamine is optionally isolated by washing the free monosaccharide (e.g., through a defined MW cut off membrane or by any other suitable method). In some of such embodiments, the free monosaccharide for detection and/or quantitation is present in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for monosaccharide content by any suitable analytical technique (e.g., HPLC, MS, GC, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS VI disease, measure disease severity, or to measure response to therapy.

MPS VII is a human genetic disease caused by a deficiency in the lysosomal enzyme beta-glucuronidase. This enzyme is required in the lysosome to degrade glycans that contain glucuronic acid residues. Due to this enzymatic deficiency, glycans with glucuronic acid residues on the non-reducing end accumulate to high levels (including chondroitin sulfate, heparan sulfate and others). In certain embodiments, using the method described herein, MPS VII is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer to remove free glucuronic acid), and treated with a glucuronidase (e.g., to liberate a glycan residual compound glucuronic acid). In some embodiments, after incubation, the liberated monosaccharide is optionally isolated by washing the free monosaccharide (e.g., through a defined MW cut off membrane or by any other suitable method). In some of such embodiments, the free monosaccharide for detection and/or quantitation is present in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for monosaccharide content by any suitable analytical technique (e.g., HPLC, MS, GC, pH detection, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect MPS VII disease, measure disease severity, or to measure response to therapy.

Methods described herein can also be used to define the relative presence of different glycan classes.

Fabry Disease is a human genetic disease caused by a deficiency in the lysosomal α-galactosidase. Due to this enzymatic deficiency, glycans with non-reducing end terminal α-galactose residues are abundant. In certain embodiments, using the method described herein, Fabry Disease is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer to remove free monosaccharide), and treated with a galactosidase that is capable of liberating a non-reducing end monosaccharide (e.g., to liberate a glycan residual compound). In some embodiments, after incubation, the liberated glycan residual compound is optionally isolated by washing the free glycan residual compound (e.g., through a defined MW cut off membrane or by any other suitable method). In some of such embodiments, the free glycan residual compound for detection and/or quantitation is present in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for glycan residual compound content by any suitable analytical technique (e.g., HPLC, MS, GC, pH detection, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect Fabry Disease, measure disease severity, or to measure response to therapy.

In some embodiments, as described in Table 1, other enzymes and processes are optionally utilized to diagnose other lysosomal storage diseases (LSDs). As described in the table, the appropriate enzyme(s) can be selected as appropriate for the specific disease.

Oncology—Melanoma and Neuroblastoma via Sialic Acid

A hallmark of cancer is altered glycosylation. The changes in glycosylation are a reflection of changes in enzymes and factors that regulate the biosynthesis, turnover, presentation, stability, solubility, and degradation of glycans. Many of these changes result in glycans being produced that have altered structures. The methods described here are utilized in various embodiments to evaluate those structural changes (e.g., measure abnormal glycan accumulation) that are present on the non-reducing end of the glycans present in individuals suffering from a cancerous disease.

Some examples of cancerous diseases suitable for diagnosis and/or monitoring therapy according to methods described herein include, by way of non-limiting example, melanoma and neuroblastoma. In some instances, such cancers have alterations in the biosynthesis, turnover, presentation, stability, solubility, or degradation of gangliosides. In some instances, these sialic acid modified glycolipids are detected and/or otherwise characterized or analyzed in a biological sample (e.g., serum) of patients with these tumor types. In some embodiments, the abundance of the heterogeneous population of gangliosides is quantified to measuring sialic acid or other glycan residual released from gangliosides in the blood.

Due to this enzymatic alteration, gangliosides and other glycans are present in the body at high levels. In certain embodiments, using the method described herein, cancer (e.g., melanoma or neuroblastoma) is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer to remove free sialic acid), and treated with a sialidase that can liberate sialic acid (e.g., to liberate a glycan residual compound sialic acid). In some embodiments, after incubation, the liberated sialic acid is optionally isolated by washing the free sialic acid (e.g., through a defined MW cut off membrane or by any other suitable method). In some of such embodiments, the free sialic acid for detection and/or quantitation is present in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for sialic acid content by any suitable analytical technique (e.g., HPLC, MS, GC, pH detection, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect cancer (e.g., melanoma or neuroblastoma) disease, measure disease severity, or to measure response to therapy.

Oncology—Myeloma via Heparan Sulfate Nonreducing ends

An example of a human cancer that is diagnosed and/or monitored according to the methods described herein (i.e., by analyzing with such a method the altered degradation of a glycan) is multiple myeloma. In certain instances, multiple myeloma commonly produces heparanase. Heparanase is an endoglycosidase that cleaved heparan sulfate into smaller fragments, exposing novel non-reducing end structures. In certain embodiments described herein, the presence of these novel non-reducing end structures are detected using any method described herein (e.g., by incubating a biological sample with various glycosidases or sulfatases to detect the presence of novel glycan non-reducing ends).

Due to this enzymatic alteration, glycans (including heparan sulfate and others) are present in the body at high levels. In certain embodiments, using the method described herein, cancer (e.g., multiple myeloma) is diagnosed in an individual from a biological sample taken therefrom. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer to remove free monosaccharides and/or sulfate), and treated with a sulfatase, iduronidase, glucuronidase, hexosaminidase, or lyase that is capable of liberating a non-reducing end monosaccharide or sulfate. In some embodiments, after incubation, the liberated glycan residual compound is optionally isolated by washing the free glycan residual compound (e.g., through a defined MW cut off membrane or by any other suitable method). In some of such embodiments, the free glycan residual compound for detection and/or quantitation is present in the flow through. In certain embodiments, the resulting isolated solution is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for glycan residual compound content by any suitable analytical technique (e.g., HPLC, MS, GC, pH detection, or the like with or without chemical or enzymatic derivatization before detection). This method can be used to detect cancer (e.g., multiple myeloma) disease, measure disease severity, or to measure response to therapy.

Oncology—Adenocarcinoma

Adenocarcinoma is associated with changes in glycosylation including increased sialylation and fucosylation. The described method can be used to measure disease by analyzing glycans (total or purified or enriched for specific glycan classes) from a patient for the amount of nonreducing end terminal sialic acid or fucose, by measuring the release of these glycan residuals after treatment with a sialidase or fucosidase.

Other Applications

As described in Tables 1-4, various diseases associated with changes in glycosylation are optionally diagnosed and/or monitored according to methods described herein. Various disorders include, by way of non-limiting example, lysosomal storage disease, cancer, neurological disease (dementia, Alzheimer's, etc), liver disease, bone disease, infectious diseases, and the like.

Provided herein are methods of diagnosing individuals (including, e.g., a disease state or the severity of a disease states) with a lysosomal storage disease (LSD) or methods of monitoring the treatment of a lysosomal storage disease (LSD). Provided in Table 1 are specific embodiments of disease that are optionally diagnosed and/or monitored according to various embodiments described herein. Table 1 also illustrates various non-limiting embodiments of specific enzyme(s) that are optionally utilized to treat a biological sample from an individual suffering from or suspected (e.g., through a pre- or preliminary screening process) of suffering from an LSD. Moreover, Table 1 further illustrates various glycan residual compounds that are liberated in various embodiments described herein, such liberated glycan residual compounds optionally being detected and/or measured in order to diagnose and/or monitor a lysosomal storage disease (LSD).

TABLE 1 Exemplary LSD Uses Glycan Non-Reducing End Primary Releasing Secondary Residual Disease Structure Enzyme Releasing Enzyme Compound MPS I IdoA iduronidase IdoA MPS II IdoA-2-O sufate 2-sulfatase Sulfate and GlcA-2-O sufate MPS II IdoA-2-O sufate 2-sulfatase Iduronidase and/or IdoA and/or and GlcA-2-O glucuronidase GlcA sufate MPS IIIA GlcN-N-sulfate N-sulfatase Sulfate MPS IIIA GlcN-N-sulfate N-sulfatase hexosaminidase GlcN MPS IIIA GlcN-N-sulfate N-sulfatase Heparin lyase GlcN MPS IIIA GlcN-N-sulfate N-sulfatase N-acetyl transferase GlcNAc and hexosaminidase MPS IIIA GlcN-N-sulfate Heparin lyase GlcN-N-sulfate MPS IIIB GlcNAc hexosaminidase GlcNAc MPS IIIB GlcNAc Deacetylase acetate MPS IIIB GlcNAc Heparin lyase GlcNAc MPS IIIC GlcNAc-6-O sulfate 6-O sulfatase Sulfate MPS IIIC GlcNAc-6-O sulfate 6-O sulfatase hexosaminidase GlcNAc MPS IIIC GlcNAc-6-O sulfate 6-O sulfatase Heparin lyase GlcNAc MPS IIIC GlcNAc-6-O sulfate Heparin lyase GlcNAc-6-O sulfate MPS IIID GlcN hexosaminidase GlcN MPS IIID GlcN Heparin lyase GlcN MPS IIID GlcN N-acetyl transferase hexosaminidase GlcNAc MPS IVA Gal-6-O sulfate and 6-O sulfatase Sulfate GalNAc-6-O sulfate MPS IVA Gal-6-O sulfate and galactosidase Gal-6-O sulfate GalNAc-6-O sulfate MPS IVA Gal-6-O sulfate and N-acetyl GalNAc-6-O GalNAc-6-O sulfate galactosidase sulfate MPS IVA Gal-6-O sulfate and hexosaminidase GalNAc-6-O GalNAc-6-O sulfate sulfate MPS IVA Gal-6-O sulfate and 6-O sulfatase galactosidase Gal GalNAc-6-O sulfate MPS IVA Gal-6-O sulfate and 6-O sulfatase N-acetyl GalNAc GalNAc-6-O sulfate galactosidase MPS IVB Gal Galactosidase Gal MPS VI GalNAc-4-O sulfate 4-O sulfatase Sulfate MPS VI GalNAc-4-O sulfate 4-O sulfatase hexosaminidase GalNAc MPS VI GalNAc-4-O sulfate 4-O sulfatase Chondroitin lyase GalNAc MPS VI GalNAc-4-O sulfate Chondroitin lyase GalNAc-4-O sulfate MPS VII GlcA β-glucuronidase GlcA Alpha Mannosidosis Mannose Manosidase Man Aspartylglucosaminuria GlcNAc hexosaminidase GlcNAc Fabry Galactose galactosidase Gal Fucosidosis Fucose fucosidase Fuc Galactosialidosis Galactose and/or Galactosidase Gal and/or Sialic acid and/or sialidase Sialic acid Gaucher glucose glucosidase glucose GM1 gangliosidosis Beta-Galactose Beta-Galactosidase galactose GM1 gangliosidosis Beta-Galactose Beta-Galactosidase Hexosaminidase GalNAc GM2 activator GalNAc hexosaminidase GalNAc deficiency Sialidosis Sialic acid Sialidase Sialic acid Sialidosis Sialic acid Alpha 2,3 Sialidase Sialic acid Sialidosis Sialic acid Alphas 2,6 Sialic acid Sialidase Sialidosis Sialic acid Alphas 2,8 Sialic acid Sialidase Krabbe Galactose galactosidase Galactose Metachromatic Sulfated 3-O sulfatase Sulfate Leukodystrophy galactosylceramide Metachromatic Sulfated 3-O sulfatase galactosidase Galactose Leukodystrophy galactosylceramide Mucolipidosis II Broad range of Any listed enzyme Any glycans monosaccharide or sulfate Mucolipidosis III Broad range of Any listed enzyme Any glycans monosaccharide or sulfate Mucolipidosis IV Broad range of Any listed enzyme Any glycans monosaccharide or sulfate Multiple Sulfatase Sulfated glycans sulfatase sulfate Deficiency Multiple Sulfatase Sulfated glycans sulfatase Any glycosidase monosaccharide Deficiency Multiple Sulfatase Sulfated glycans Any glycosidase Sulfated Deficiency monosaccharide Glycogen Storage glucose glucosidase glucose Disease (Pompe) Sandhoff GalNAc hexosaminidase GalNAc Tay-Sachs GalNAc hexosaminidase GalNAc AB Variant GalNAc hexosaminidase GalNAc Schindler Disease Alpha-GalNAc hexosaminidase GalNAc Salla Disease Sialic acid none Sialic Acid Alpha Mannosidosis Alpha mannose mannosidase Mannose Beta Mannosidosis Beta mannose mannosidase Mannose Globoid cell galactose galactosidase galactose leukodystrophy

Provided herein are methods of diagnosing individuals (including, e.g., a disease state or the severity of a disease states) with a cancerous disease state or methods of monitoring the treatment of a cancer. Provided in Table 2 are specific embodiments of disease that are optionally diagnosed and/or monitored according to various embodiments described herein. Table 2 also illustrates various non-limiting embodiments of specific enzyme(s) that are optionally utilized to treat a biological sample from an individual suffering from or suspected of (e.g., through a pre- or preliminary screening process) suffering from a cancerous disease state. Moreover, Table 2 further illustrates various glycan residual compounds that are liberated in various embodiments described herein, such liberated glycan residual compounds optionally being detected and/or measured in order to diagnose and/or monitor a cancerous disease state.

TABLE 2 Exemplary Oncology Uses Secondary Glycan Non-Reducing Liberating Residual Cancer Type End Structure Primary Liberating Enzyme Enzyme Compound Melanoma Sialic Acid Sialidase Sialic acid Melanoma Sialic Acid Alpha 2,8 Sialidase Sialic acid Melanoma Sialic Acid Alpha 2,3 Sialidase Sialic acid Melanoma Sialic Acid Alpha 2,6 Sialidase Sialic acid Melanoma GalNAc Hexosaminidase GalNAc Melanoma GalNAc Sialidase Hexosaminidase GalNAc Melanoma Sialic acid Hexosaminidase Sialidase Sialic acid Melanoma Galactose galactosidase Galactose Melanoma Galactose sialidase galactosidase Galactose Melanoma Fucose fucosidase Fucose Melanoma Galactose Galactosidase Galactose Melanoma GlcNAc hexosaminidase GlcNAc Melanoma Sulfate Sulfatase Sulfate Melanoma Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Melanoma Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Neuroblastoma Sialic Acid Sialidase Sialic acid Neuroblastoma Sialic Acid Alpha 2,8 Sialidase Sialic acid Neuroblastoma Sialic Acid Alpha 2,3 Sialidase Sialic acid Neuroblastoma Sialic Acid Alpha 2,6 Sialidase Sialic acid Neuroblastoma GalNAc Hexosaminidase GalNAc Neuroblastoma GalNAc Sialidase Hexosaminidase GalNAc Neuroblastoma Sialic acid Hexosaminidase Sialidase Sialic acid Neuroblastoma Galactose galactosidase Galactose Neuroblastoma Galactose sialidase galactosidase Galactose Neuroblastoma Fucose fucosidase Fucose Neuroblastoma Galactose Galactosidase Galactose Neuroblastoma GlcNAc hexosaminidase GlcNAc Neuroblastoma Sulfate Sulfatase Sulfate Neuroblastoma Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Neuroblastoma Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Adenocarcinoma Sialic Acid Sialidase Sialic acid Adenocarcinoma Sialic Acid Alpha 2,8 Sialidase Sialic acid Adenocarcinoma Sialic Acid Alpha 2,3 Sialidase Sialic acid Adenocarcinoma Sialic Acid Alpha 2,6 Sialidase Sialic acid Adenocarcinoma GalNAc Hexosaminidase GalNAc Adenocarcinoma GalNAc Sialidase Hexosaminidase GalNAc Adenocarcinoma Sialic acid Hexosaminidase Sialidase Sialic acid Adenocarcinoma Galactose galactosidase Galactose Adenocarcinoma Galactose sialidase galactosidase Galactose Adenocarcinoma Fucose fucosidase Fucose Adenocarcinoma Galactose Galactosidase Galactose Adenocarcinoma GlcNAc hexosaminidase GlcNAc Adenocarcinoma Sulfate Sulfatase Sulfate Adenocarcinoma Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Adenocarcinoma Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Myeloma Sialic Acid Sialidase Sialic acid Myeloma Sialic Acid Alpha 2,8 Sialidase Sialic acid Myeloma Sialic Acid Alpha 2,3 Sialidase Sialic acid Myeloma Sialic Acid Alpha 2,6 Sialidase Sialic acid Myeloma GalNAc Hexosaminidase GalNAc Myeloma GalNAc Sialidase Hexosaminidase GalNAc Myeloma Sialic acid Hexosaminidase Sialidase Sialic acid Myeloma Galactose galactosidase Galactose Myeloma Galactose sialidase galactosidase Galactose Myeloma Fucose fucosidase Fucose Myeloma Galactose Galactosidase Galactose Myeloma GlcNAc hexosaminidase GlcNAc Myeloma Sulfate Sulfatase Sulfate Myeloma Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Myeloma Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Breast Sialic Acid Sialidase Sialic acid Breast Sialic Acid Alpha 2,8 Sialidase Sialic acid Breast Sialic Acid Alpha 2,3 Sialidase Sialic acid Breast Sialic Acid Alpha 2,6 Sialidase Sialic acid Breast GalNAc Hexosaminidase GalNAc Breast GalNAc Sialidase Hexosaminidase GalNAc Breast Sialic acid Hexosaminidase Sialidase Sialic acid Breast Galactose galactosidase Galactose Breast Galactose sialidase galactosidase Galactose Breast Fucose fucosidase Fucose Breast Galactose Galactosidase Galactose Breast GlcNAc hexosaminidase GlcNAc Breast Sulfate Sulfatase Sulfate Breast Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Breast Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Ovarian Sialic Acid Sialidase Sialic acid Ovarian Sialic Acid Alpha 2,8 Sialidase Sialic acid Ovarian Sialic Acid Alpha 2,3 Sialidase Sialic acid Ovarian Sialic Acid Alpha 2,6 Sialidase Sialic acid Ovarian GalNAc Hexosaminidase GalNAc Ovarian GalNAc Sialidase Hexosaminidase GalNAc Ovarian Sialic acid Hexosaminidase Sialidase Sialic acid Ovarian Galactose galactosidase Galactose Ovarian Galactose sialidase galactosidase Galactose Ovarian Fucose fucosidase Fucose Ovarian Galactose Galactosidase Galactose Ovarian GlcNAc hexosaminidase GlcNAc Ovarian Sulfate Sulfatase Sulfate Ovarian Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Ovarian Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Stomach Sialic Acid Sialidase Sialic acid Stomach Sialic Acid Alpha 2,8 Sialidase Sialic acid Stomach Sialic Acid Alpha 2,3 Sialidase Sialic acid Stomach Sialic Acid Alpha 2,6 Sialidase Sialic acid Stomach GalNAc Hexosaminidase GalNAc Stomach GalNAc Sialidase Hexosaminidase GalNAc Stomach Sialic acid Hexosaminidase Sialidase Sialic acid Stomach Galactose galactosidase Galactose Stomach Galactose sialidase galactosidase Galactose Stomach Fucose fucosidase Fucose Stomach Galactose Galactosidase Galactose Stomach GlcNAc hexosaminidase GlcNAc Stomach Sulfate Sulfatase Sulfate Stomach Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Stomach Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Lung Sialic Acid Sialidase Sialic acid Lung Sialic Acid Alpha 2,8 Sialidase Sialic acid Lung Sialic Acid Alpha 2,3 Sialidase Sialic acid Lung Sialic Acid Alpha 2,6 Sialidase Sialic acid Lung GalNAc Hexosaminidase GalNAc Lung GalNAc Sialidase Hexosaminidase GalNAc Lung Sialic acid Hexosaminidase Sialidase Sialic acid Lung Galactose galactosidase Galactose Lung Galactose sialidase galactosidase Galactose Lung Fucose fucosidase Fucose Lung Galactose Galactosidase Galactose Lung GlcNAc hexosaminidase GlcNAc Lung Sulfate Sulfatase Sulfate Lung Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Lung Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Pancreatic Sialic Acid Sialidase Sialic acid Pancreatic Sialic Acid Alpha 2,8 Sialidase Sialic acid Pancreatic Sialic Acid Alpha 2,3 Sialidase Sialic acid Pancreatic Sialic Acid Alpha 2,6 Sialidase Sialic acid Pancreatic GalNAc Hexosaminidase GalNAc Pancreatic GalNAc Sialidase Hexosaminidase GalNAc Pancreatic Sialic acid Hexosaminidase Sialidase Sialic acid Pancreatic Galactose galactosidase Galactose Pancreatic Galactose sialidase galactosidase Galactose Pancreatic Fucose fucosidase Fucose Pancreatic Galactose Galactosidase Galactose Pancreatic GlcNAc hexosaminidase GlcNAc Pancreatic Sulfate Sulfatase Sulfate Pancreatic Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Pancreatic Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Oral Sialic Acid Sialidase Sialic acid Oral Sialic Acid Alpha 2,8 Sialidase Sialic acid Oral Sialic Acid Alpha 2,3 Sialidase Sialic acid Oral Sialic Acid Alpha 2,6 Sialidase Sialic acid Oral GalNAc Hexosaminidase GalNAc Oral GalNAc Sialidase Hexosaminidase GalNAc Oral Sialic acid Hexosaminidase Sialidase Sialic acid Oral Galactose galactosidase Galactose Oral Galactose sialidase galactosidase Galactose Oral Fucose fucosidase Fucose Oral Galactose Galactosidase Galactose Oral GlcNAc hexosaminidase GlcNAc Oral Sulfate Sulfatase Sulfate Oral Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Oral Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Colorectal Sialic Acid Sialidase Sialic acid Colorectal Sialic Acid Alpha 2,8 Sialidase Sialic acid Colorectal Sialic Acid Alpha 2,3 Sialidase Sialic acid Colorectal Sialic Acid Alpha 2,6 Sialidase Sialic acid Colorectal GalNAc Hexosaminidase GalNAc Colorectal GalNAc Sialidase Hexosaminidase GalNAc Colorectal Sialic acid Hexosaminidase Sialidase Sialic acid Colorectal Galactose galactosidase Galactose Colorectal Galactose sialidase galactosidase Galactose Colorectal Fucose fucosidase Fucose Colorectal Galactose Galactosidase Galactose Colorectal GlcNAc hexosaminidase GlcNAc Colorectal Sulfate Sulfatase Sulfate Colorectal Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Colorectal Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Kidney Sialic Acid Sialidase Sialic acid Kidney Sialic Acid Alpha 2,8 Sialidase Sialic acid Kidney Sialic Acid Alpha 2,3 Sialidase Sialic acid Kidney Sialic Acid Alpha 2,6 Sialidase Sialic acid Kidney GalNAc Hexosaminidase GalNAc Kidney GalNAc Sialidase Hexosaminidase GalNAc Kidney Sialic acid Hexosaminidase Sialidase Sialic acid Kidney Galactose galactosidase Galactose Kidney Galactose sialidase galactosidase Galactose Kidney Fucose fucosidase Fucose Kidney Galactose Galactosidase Galactose Kidney GlcNAc hexosaminidase GlcNAc Kidney Sulfate Sulfatase Sulfate Kidney Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Kidney Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Bladder Sialic Acid Sialidase Sialic acid Bladder Sialic Acid Alpha 2,8 Sialidase Sialic acid Bladder Sialic Acid Alpha 2,3 Sialidase Sialic acid Bladder Sialic Acid Alpha 2,6 Sialidase Sialic acid Bladder GalNAc Hexosaminidase GalNAc Bladder GalNAc Sialidase Hexosaminidase GalNAc Bladder Sialic acid Hexosaminidase Sialidase Sialic acid Bladder Galactose galactosidase Galactose Bladder Galactose sialidase galactosidase Galactose Bladder Fucose fucosidase Fucose Bladder Galactose Galactosidase Galactose Bladder GlcNAc hexosaminidase GlcNAc Bladder Sulfate Sulfatase Sulfate Bladder Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Bladder Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Prostate Sialic Acid Sialidase Sialic acid Prostate Sialic Acid Alpha 2,8 Sialidase Sialic acid Prostate Sialic Acid Alpha 2,3 Sialidase Sialic acid Prostate Sialic Acid Alpha 2,6 Sialidase Sialic acid Prostate GalNAc Hexosaminidase GalNAc Prostate GalNAc Sialidase Hexosaminidase GalNAc Prostate Sialic acid Hexosaminidase Sialidase Sialic acid Prostate Galactose galactosidase Galactose Prostate Galactose sialidase galactosidase Galactose Prostate Fucose fucosidase Fucose Prostate Galactose Galactosidase Galactose Prostate GlcNAc hexosaminidase GlcNAc Prostate Sulfate Sulfatase Sulfate Prostate Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Prostate Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Uterine Sialic Acid Sialidase Sialic acid Uterine Sialic Acid Alpha 2,8 Sialidase Sialic acid Uterine Sialic Acid Alpha 2,3 Sialidase Sialic acid Uterine Sialic Acid Alpha 2,6 Sialidase Sialic acid Uterine GalNAc Hexosaminidase GalNAc Uterine GalNAc Sialidase Hexosaminidase GalNAc Uterine Sialic acid Hexosaminidase Sialidase Sialic acid Uterine Galactose galactosidase Galactose Uterine Galactose sialidase galactosidase Galactose Uterine Fucose fucosidase Fucose Uterine Galactose Galactosidase Galactose Uterine GlcNAc hexosaminidase GlcNAc Uterine Sulfate Sulfatase Sulfate Uterine Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Uterine Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Thyroid Sialic Acid Sialidase Sialic acid Thyroid Sialic Acid Alpha 2,8 Sialidase Sialic acid Thyroid Sialic Acid Alpha 2,3 Sialidase Sialic acid Thyroid Sialic Acid Alpha 2,6 Sialidase Sialic acid Thyroid GalNAc Hexosaminidase GalNAc Thyroid GalNAc Sialidase Hexosaminidase GalNAc Thyroid Sialic acid Hexosaminidase Sialidase Sialic acid Thyroid Galactose galactosidase Galactose Thyroid Galactose sialidase galactosidase Galactose Thyroid Fucose fucosidase Fucose Thyroid Galactose Galactosidase Galactose Thyroid GlcNAc hexosaminidase GlcNAc Thyroid Sulfate Sulfatase Sulfate Thyroid Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Thyroid Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Liver Sialic Acid Sialidase Sialic acid Liver Sialic Acid Alpha 2,8 Sialidase Sialic acid Liver Sialic Acid Alpha 2,3 Sialidase Sialic acid Liver Sialic Acid Alpha 2,6 Sialidase Sialic acid Liver GalNAc Hexosaminidase GalNAc Liver GalNAc Sialidase Hexosaminidase GalNAc Liver Sialic acid Hexosaminidase Sialidase Sialic acid Liver Galactose galactosidase Galactose Liver Galactose sialidase galactosidase Galactose Liver Fucose fucosidase Fucose Liver Galactose Galactosidase Galactose Liver GlcNAc hexosaminidase GlcNAc Liver Sulfate Sulfatase Sulfate Liver Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Liver Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Esophagus Sialic Acid Sialidase Sialic acid Esophagus Sialic Acid Alpha 2,8 Sialidase Sialic acid Esophagus Sialic Acid Alpha 2,3 Sialidase Sialic acid Esophagus Sialic Acid Alpha 2,6 Sialidase Sialic acid Esophagus GalNAc Hexosaminidase GalNAc Esophagus GalNAc Sialidase Hexosaminidase GalNAc Esophagus Sialic acid Hexosaminidase Sialidase Sialic acid Esophagus Galactose galactosidase Galactose Esophagus Galactose sialidase galactosidase Galactose Esophagus Fucose fucosidase Fucose Esophagus Galactose Galactosidase Galactose Esophagus GlcNAc hexosaminidase GlcNAc Esophagus Sulfate Sulfatase Sulfate Esophagus Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Esophagus Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Brain Sialic Acid Sialidase Sialic acid Brain Sialic Acid Alpha 2,8 Sialidase Sialic acid Brain Sialic Acid Alpha 2,3 Sialidase Sialic acid Brain Sialic Acid Alpha 2,6 Sialidase Sialic acid Brain GalNAc Hexosaminidase GalNAc Brain GalNAc Sialidase Hexosaminidase GalNAc Brain Sialic acid Hexosaminidase Sialidase Sialic acid Brain Galactose galactosidase Galactose Brain Galactose sialidase galactosidase Galactose Brain Fucose fucosidase Fucose Brain Galactose Galactosidase Galactose Brain GlcNAc hexosaminidase GlcNAc Brain Sulfate Sulfatase Sulfate Brain Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Brain Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Lymphomas Sialic Acid Sialidase Sialic acid Lymphomas Sialic Acid Alpha 2,8 Sialidase Sialic acid Lymphomas Sialic Acid Alpha 2,3 Sialidase Sialic acid Lymphomas Sialic Acid Alpha 2,6 Sialidase Sialic acid Lymphomas GalNAc Hexosaminidase GalNAc Lymphomas GalNAc Sialidase Hexosaminidase GalNAc Lymphomas Sialic acid Hexosaminidase Sialidase Sialic acid Lymphomas Galactose galactosidase Galactose Lymphomas Galactose sialidase galactosidase Galactose Lymphomas Fucose fucosidase Fucose Lymphomas Galactose Galactosidase Galactose Lymphomas GlcNAc hexosaminidase GlcNAc Lymphomas Sulfate Sulfatase Sulfate Lymphomas Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Lymphomas Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA Leukemias Sialic Acid Sialidase Sialic acid Leukemias Sialic Acid Alpha 2,8 Sialidase Sialic acid Leukemias Sialic Acid Alpha 2,3 Sialidase Sialic acid Leukemias Sialic Acid Alpha 2,6 Sialidase Sialic acid Leukemias GalNAc Hexosaminidase GalNAc Leukemias GalNAc Sialidase Hexosaminidase GalNAc Leukemias Sialic acid Hexosaminidase Sialidase Sialic acid Leukemias Galactose galactosidase Galactose Leukemias Galactose sialidase galactosidase Galactose Leukemias Fucose fucosidase Fucose Leukemias Galactose Galactosidase Galactose Leukemias GlcNAc hexosaminidase GlcNAc Leukemias Sulfate Sulfatase Sulfate Leukemias Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Leukemias Sulfated uronic Sulfatase Iduronidase or IdoA or acid glucouronidase GlcA

Provided herein are methods of diagnosing individuals (including, e.g., a disease state or the severity of a disease states) with a disease state associated with abnormal glycan accumulation. Provided in Table 3 are specific embodiments of disease that are optionally diagnosed and/or monitored according to various embodiments described herein. Table 3 also illustrates various non-limiting embodiments of specific enzyme(s) that are optionally utilized to treat a biological sample from an individual suffering from or suspected of (e.g., through a pre- or preliminary screening process) suffering from various disease states associated with abnormal glycan accumulation. Moreover, Table 3 further illustrates various glycan residual compounds that are liberated in various embodiments described herein, such liberated glycan residual compounds optionally being detected and/or measured in order to diagnose and/or monitor various disease states.

TABLE 3 Primary Glycan Non-Reducing Liberating Secondary Residual Disease End Structure Enzyme Liberating Enzyme Compound Alzheimers Sialic Acid Sialidase Sialic acid Alzheimers Sialic Acid Alpha 2,8 Sialidase Sialic acid Alzheimers Sialic Acid Alpha 2,3 Sialidase Sialic acid Alzheimers Sialic Acid Alpha 2,6 Sialidase Sialic acid Alzheimers GalNAc Hexosaminidase GalNAc Alzheimers GalNAc Sialidase Hexosaminidase GalNAc Alzheimers Sialic acid Hexosaminidase Sialidase Sialic acid Alzheimers Galactose galactosidase Galactose Alzheimers Galactose sialidase galactosidase Galactose Alzheimers Fucose fucosidase Fucose Alzheimers Galactose Galactosidase Galactose Alzheimers GlcNAc hexosaminidase GlcNAc Alzheimers Sulfate Sulfatase Sulfate Alzheimers Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Alzheimers Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucuronidase Amyotrophic Lateral Sialic Acid Sialidase Sialic acid Sclerosis Amyotrophic Lateral Sialic Acid Alpha 2,8 Sialidase Sialic acid Sclerosis Amyotrophic Lateral Sialic Acid Alpha 2,3 Sialidase Sialic acid Sclerosis Amyotrophic Lateral Sialic Acid Alpha 2,6 Sialidase Sialic acid Sclerosis Amyotrophic Lateral GalNAc Hexosaminidase GalNAc Sclerosis Amyotrophic Lateral GalNAc Sialidase Hexosaminidase GalNAc Sclerosis Amyotrophic Lateral Sialic acid Hexosaminidase Sialidase Sialic acid Sclerosis Amyotrophic Lateral Galactose galactosidase Galactose Sclerosis Amyotrophic Lateral Galactose sialidase galactosidase Galactose Sclerosis Amyotrophic Lateral Fucose fucosidase Fucose Sclerosis Amyotrophic Lateral Galactose Galactosidase Galactose Sclerosis Amyotrophic Lateral GlcNAc hexosaminidase GlcNAc Sclerosis Amyotrophic Lateral Sulfate Sulfatase Sulfate Sclerosis Amyotrophic Lateral Sulfated hexose Sulfatase hexosaminidase GlcNAc or Sclerosis GalNAc Amyotrophic Lateral Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA Sclerosis acid glucuronidase Cerebral Palsy Sialic Acid Sialidase Sialic acid Cerebral Palsy Sialic Acid Alpha 2,8 Sialidase Sialic acid Cerebral Palsy Sialic Acid Alpha 2,3 Sialidase Sialic acid Cerebral Palsy Sialic Acid Alpha 2,6 Sialidase Sialic acid Cerebral Palsy GalNAc Hexosaminidase GalNAc Cerebral Palsy GalNAc Sialidase Hexosaminidase GalNAc Cerebral Palsy Sialic acid Hexosaminidase Sialidase Sialic acid Cerebral Palsy Galactose galactosidase Galactose Cerebral Palsy Galactose sialidase galactosidase Galactose Cerebral Palsy Fucose fucosidase Fucose Cerebral Palsy Galactose Galactosidase Galactose Cerebral Palsy GlcNAc hexosaminidase GlcNAc Cerebral Palsy Sulfate Sulfatase Sulfate Cerebral Palsy Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Cerebral Palsy Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucuronidase Schizophrenia Sialic Acid Sialidase Sialic acid Schizophrenia Sialic Acid Alpha 2,8 Sialidase Sialic acid Schizophrenia Sialic Acid Alpha 2,3 Sialidase Sialic acid Schizophrenia Sialic Acid Alpha 2,6 Sialidase Sialic acid Schizophrenia GalNAc Hexosaminidase GalNAc Schizophrenia GalNAc Sialidase Hexosaminidase GalNAc Schizophrenia Sialic acid Hexosaminidase Sialidase Sialic acid Schizophrenia Galactose galactosidase Galactose Schizophrenia Galactose sialidase galactosidase Galactose Schizophrenia Fucose fucosidase Fucose Schizophrenia Galactose Galactosidase Galactose Schizophrenia GlcNAc hexosaminidase GlcNAc Schizophrenia Sulfate Sulfatase Sulfate Schizophrenia Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Schizophrenia Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Bipolar Disorder Sialic Acid Sialidase Sialic acid Bipolar Disorder Sialic Acid Alpha 2,8 Sialidase Sialic acid Bipolar Disorder Sialic Acid Alpha 2,3 Sialidase Sialic acid Bipolar Disorder Sialic Acid Alpha 2,6 Sialidase Sialic acid Bipolar Disorder GalNAc Hexosaminidase GalNAc Bipolar Disorder GalNAc Sialidase Hexosaminidase GalNAc Bipolar Disorder Sialic acid Hexosaminidase Sialidase Sialic acid Bipolar Disorder Galactose galactosidase Galactose Bipolar Disorder Galactose sialidase galactosidase Galactose Bipolar Disorder Fucose fucosidase Fucose Bipolar Disorder Galactose Galactosidase Galactose Bipolar Disorder GlcNAc hexosaminidase GlcNAc Bipolar Disorder Sulfate Sulfatase Sulfate Bipolar Disorder Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Bipolar Disorder Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Depression Sialic Acid Sialidase Sialic acid Depression Sialic Acid Alpha 2,8 Sialidase Sialic acid Depression Sialic Acid Alpha 2,3 Sialidase Sialic acid Depression Sialic Acid Alpha 2,6 Sialidase Sialic acid Depression GalNAc Hexosaminidase GalNAc Depression GalNAc Sialidase Hexosaminidase GalNAc Depression Sialic acid Hexosaminidase Sialidase Sialic acid Depression Galactose galactosidase Galactose Depression Galactose sialidase galactosidase Galactose Depression Fucose fucosidase Fucose Depression Galactose Galactosidase Galactose Depression GlcNAc hexosaminidase GlcNAc Depression Sulfate Sulfatase Sulfate Depression Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Depression Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Epilepsy Sialic Acid Sialidase Sialic acid Epilepsy Sialic Acid Alpha 2,8 Sialidase Sialic acid Epilepsy Sialic Acid Alpha 2,3 Sialidase Sialic acid Epilepsy Sialic Acid Alpha 2,6 Sialidase Sialic acid Epilepsy GalNAc Hexosaminidase GalNAc Epilepsy GalNAc Sialidase Hexosaminidase GalNAc Epilepsy Sialic acid Hexosaminidase Sialidase Sialic acid Epilepsy Galactose galactosidase Galactose Epilepsy Galactose sialidase galactosidase Galactose Epilepsy Fucose fucosidase Fucose Epilepsy Galactose Galactosidase Galactose Epilepsy GlcNAc hexosaminidase GlcNAc Epilepsy Sulfate Sulfatase Sulfate Epilepsy Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Epilepsy Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Migraine Sialic Acid Sialidase Sialic acid Migraine Sialic Acid Alpha 2,8 Sialidase Sialic acid Migraine Sialic Acid Alpha 2,3 Sialidase Sialic acid Migraine Sialic Acid Alpha 2,6 Sialidase Sialic acid Migraine GalNAc Hexosaminidase GalNAc Migraine GalNAc Sialidase Hexosaminidase GalNAc Migraine Sialic acid Hexosaminidase Sialidase Sialic acid Migraine Galactose galactosidase Galactose Migraine Galactose sialidase galactosidase Galactose Migraine Fucose fucosidase Fucose Migraine Galactose Galactosidase Galactose Migraine GlcNAc hexosaminidase GlcNAc Migraine Sulfate Sulfatase Sulfate Migraine Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Migraine Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Multiple Sclerosis Sialic Acid Sialidase Sialic acid Multiple Sclerosis Sialic Acid Alpha 2,8 Sialidase Sialic acid Multiple Sclerosis Sialic Acid Alpha 2,3 Sialidase Sialic acid Multiple Sclerosis Sialic Acid Alpha 2,6 Sialidase Sialic acid Multiple Sclerosis GalNAc Hexosaminidase GalNAc Multiple Sclerosis GalNAc Sialidase Hexosaminidase GalNAc Multiple Sclerosis Sialic acid Hexosaminidase Sialidase Sialic acid Multiple Sclerosis Galactose galactosidase Galactose Multiple Sclerosis Galactose sialidase galactosidase Galactose Multiple Sclerosis Fucose fucosidase Fucose Multiple Sclerosis Galactose Galactosidase Galactose Multiple Sclerosis GlcNAc hexosaminidase GlcNAc Multiple Sclerosis Sulfate Sulfatase Sulfate Multiple Sclerosis Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Multiple Sclerosis Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Parkinson's Sialic Acid Sialidase Sialic acid Parkinson's Sialic Acid Alpha 2,8 Sialidase Sialic acid Parkinson's Sialic Acid Alpha 2,3 Sialidase Sialic acid Parkinson's Sialic Acid Alpha 2,6 Sialidase Sialic acid Parkinson's GalNAc Hexosaminidase GalNAc Parkinson's GalNAc Sialidase Hexosaminidase GalNAc Parkinson's Sialic acid Hexosaminidase Sialidase Sialic acid Parkinson's Galactose galactosidase Galactose Parkinson's Galactose sialidase galactosidase Galactose Parkinson's Fucose fucosidase Fucose Parkinson's Galactose Galactosidase Galactose Parkinson's GlcNAc hexosaminidase GlcNAc Parkinson's Sulfate Sulfatase Sulfate Parkinson's Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Parkinson's Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Rheumatoid Arthritis Sialic Acid Sialidase Sialic acid Rheumatoid Arthritis Sialic Acid Alpha 2,8 Sialidase Sialic acid Rheumatoid Arthritis Sialic Acid Alpha 2,3 Sialidase Sialic acid Rheumatoid Arthritis Sialic Acid Alpha 2,6 Sialidase Sialic acid Rheumatoid Arthritis GalNAc Hexosaminidase GalNAc Rheumatoid Arthritis GalNAc Sialidase Hexosaminidase GalNAc Rheumatoid Arthritis Sialic acid Hexosaminidase Sialidase Sialic acid Rheumatoid Arthritis Galactose galactosidase Galactose Rheumatoid Arthritis Galactose sialidase galactosidase Galactose Rheumatoid Arthritis Fucose fucosidase Fucose Rheumatoid Arthritis Galactose Galactosidase Galactose Rheumatoid Arthritis GlcNAc hexosaminidase GlcNAc Rheumatoid Arthritis Sulfate Sulfatase Sulfate Rheumatoid Arthritis Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Rheumatoid Arthritis Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Psoriatic Arthritis Sialic Acid Sialidase Sialic acid Psoriatic Arthritis Sialic Acid Alpha 2,8 Sialidase Sialic acid Psoriatic Arthritis Sialic Acid Alpha 2,3 Sialidase Sialic acid Psoriatic Arthritis Sialic Acid Alpha 2,6 Sialidase Sialic acid Psoriatic Arthritis GalNAc Hexosaminidase GalNAc Psoriatic Arthritis GalNAc Sialidase Hexosaminidase GalNAc Psoriatic Arthritis Sialic acid Hexosaminidase Sialidase Sialic acid Psoriatic Arthritis Galactose galactosidase Galactose Psoriatic Arthritis Galactose sialidase galactosidase Galactose Psoriatic Arthritis Fucose fucosidase Fucose Psoriatic Arthritis Galactose Galactosidase Galactose Psoriatic Arthritis GlcNAc hexosaminidase GlcNAc Psoriatic Arthritis Sulfate Sulfatase Sulfate Psoriatic Arthritis Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Psoriatic Arthritis Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Asthma Sialic Acid Sialidase Sialic acid Asthma Sialic Acid Alpha 2,8 Sialidase Sialic acid Asthma Sialic Acid Alpha 2,3 Sialidase Sialic acid Asthma Sialic Acid Alpha 2,6 Sialidase Sialic acid Asthma GalNAc Hexosaminidase GalNAc Asthma GalNAc Sialidase Hexosaminidase GalNAc Asthma Sialic acid Hexosaminidase Sialidase Sialic acid Asthma Galactose galactosidase Galactose Asthma Galactose sialidase galactosidase Galactose Asthma Fucose fucosidase Fucose Asthma Galactose Galactosidase Galactose Asthma GlcNAc hexosaminidase GlcNAc Asthma Sulfate Sulfatase Sulfate Asthma Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Asthma Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Chronic Obstructive Sialic Acid Sialidase Sialic acid Pulmonary Disorder Chronic Obstructive Sialic Acid Alpha 2,8 Sialidase Sialic acid Pulmonary Disorder Chronic Obstructive Sialic Acid Alpha 2,3 Sialidase Sialic acid Pulmonary Disorder Chronic Obstructive Sialic Acid Alpha 2,6 Sialidase Sialic acid Pulmonary Disorder Chronic Obstructive GalNAc Hexosaminidase GalNAc Pulmonary Disorder Chronic Obstructive GalNAc Sialidase Hexosaminidase GalNAc Pulmonary Disorder Chronic Obstructive Sialic acid Hexosaminidase Sialidase Sialic acid Pulmonary Disorder Chronic Obstructive Galactose galactosidase Galactose Pulmonary Disorder Chronic Obstructive Galactose sialidase galactosidase Galactose Pulmonary Disorder Chronic Obstructive Fucose fucosidase Fucose Pulmonary Disorder Chronic Obstructive Galactose Galactosidase Galactose Pulmonary Disorder Chronic Obstructive GlcNAc hexosaminidase GlcNAc Pulmonary Disorder Chronic Obstructive Sulfate Sulfatase Sulfate Pulmonary Disorder Chronic Obstructive Sulfated hexose Sulfatase hexosaminidase GlcNAc or Pulmonary Disorder GalNAc Chronic Obstructive Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA Pulmonary Disorder acid glucouronidase Lupus Sialic Acid Sialidase Sialic acid Lupus Sialic Acid Alpha 2,8 Sialidase Sialic acid Lupus Sialic Acid Alpha 2,3 Sialidase Sialic acid Lupus Sialic Acid Alpha 2,6 Sialidase Sialic acid Lupus GalNAc Hexosaminidase GalNAc Lupus GalNAc Sialidase Hexosaminidase GalNAc Lupus Sialic acid Hexosaminidase Sialidase Sialic acid Lupus Galactose galactosidase Galactose Lupus Galactose sialidase galactosidase Galactose Lupus Fucose fucosidase Fucose Lupus Galactose Galactosidase Galactose Lupus GlcNAc hexosaminidase GlcNAc Lupus Sulfate Sulfatase Sulfate Lupus Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Lupus Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Hepatitis Sialic Acid Sialidase Sialic acid Hepatitis Sialic Acid Alpha 2,8 Sialidase Sialic acid Hepatitis Sialic Acid Alpha 2,3 Sialidase Sialic acid Hepatitis Sialic Acid Alpha 2,6 Sialidase Sialic acid Hepatitis GalNAc Hexosaminidase GalNAc Hepatitis GalNAc Sialidase Hexosaminidase GalNAc Hepatitis Sialic acid Hexosaminidase Sialidase Sialic acid Hepatitis Galactose galactosidase Galactose Hepatitis Galactose sialidase galactosidase Galactose Hepatitis Fucose fucosidase Fucose Hepatitis Galactose Galactosidase Galactose Hepatitis GlcNAc hexosaminidase GlcNAc Hepatitis Sulfate Sulfatase Sulfate Hepatitis Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Hepatitis Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Renal Disease Sialic Acid Sialidase Sialic acid Renal Disease Sialic Acid Alpha 2,8 Sialidase Sialic acid Renal Disease Sialic Acid Alpha 2,3 Sialidase Sialic acid Renal Disease Sialic Acid Alpha 2,6 Sialidase Sialic acid Renal Disease GalNAc Hexosaminidase GalNAc Renal Disease GalNAc Sialidase Hexosaminidase GalNAc Renal Disease Sialic acid Hexosaminidase Sialidase Sialic acid Renal Disease Galactose galactosidase Galactose Renal Disease Galactose sialidase galactosidase Galactose Renal Disease Fucose fucosidase Fucose Renal Disease Galactose Galactosidase Galactose Renal Disease GlcNAc hexosaminidase GlcNAc Renal Disease Sulfate Sulfatase Sulfate Renal Disease Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Renal Disease Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Sickle Cell Disease Sialic Acid Sialidase Sialic acid Sickle Cell Disease Sialic Acid Alpha 2,8 Sialidase Sialic acid Sickle Cell Disease Sialic Acid Alpha 2,3 Sialidase Sialic acid Sickle Cell Disease Sialic Acid Alpha 2,6 Sialidase Sialic acid Sickle Cell Disease GalNAc Hexosaminidase GalNAc Sickle Cell Disease GalNAc Sialidase Hexosaminidase GalNAc Sickle Cell Disease Sialic acid Hexosaminidase Sialidase Sialic acid Sickle Cell Disease Galactose galactosidase Galactose Sickle Cell Disease Galactose sialidase galactosidase Galactose Sickle Cell Disease Fucose fucosidase Fucose Sickle Cell Disease Galactose Galactosidase Galactose Sickle Cell Disease GlcNAc hexosaminidase GlcNAc Sickle Cell Disease Sulfate Sulfatase Sulfate Sickle Cell Disease Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Sickle Cell Disease Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Fibromyalgia Sialic Acid Sialidase Sialic acid Fibromyalgia Sialic Acid Alpha 2,8 Sialidase Sialic acid Fibromyalgia Sialic Acid Alpha 2,3 Sialidase Sialic acid Fibromyalgia Sialic Acid Alpha 2,6 Sialidase Sialic acid Fibromyalgia GalNAc Hexosaminidase GalNAc Fibromyalgia GalNAc Sialidase Hexosaminidase GalNAc Fibromyalgia Sialic acid Hexosaminidase Sialidase Sialic acid Fibromyalgia Galactose galactosidase Galactose Fibromyalgia Galactose sialidase galactosidase Galactose Fibromyalgia Fucose fucosidase Fucose Fibromyalgia Galactose Galactosidase Galactose Fibromyalgia GlcNAc hexosaminidase GlcNAc Fibromyalgia Sulfate Sulfatase Sulfate Fibromyalgia Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Fibromyalgia Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Irritable Bowel Syndrome Sialic Acid Sialidase Sialic acid Irritable Bowel Syndrome Sialic Acid Alpha 2,8 Sialidase Sialic acid Irritable Bowel Syndrome Sialic Acid Alpha 2,3 Sialidase Sialic acid Irritable Bowel Syndrome Sialic Acid Alpha 2,6 Sialidase Sialic acid Irritable Bowel Syndrome GalNAc Hexosaminidase GalNAc Irritable Bowel Syndrome GalNAc Sialidase Hexosaminidase GalNAc Irritable Bowel Syndrome Sialic acid Hexosaminidase Sialidase Sialic acid Irritable Bowel Syndrome Galactose galactosidase Galactose Irritable Bowel Syndrome Galactose sialidase galactosidase Galactose Irritable Bowel Syndrome Fucose fucosidase Fucose Irritable Bowel Syndrome Galactose Galactosidase Galactose Irritable Bowel Syndrome GlcNAc hexosaminidase GlcNAc Irritable Bowel Syndrome Sulfate Sulfatase Sulfate Irritable Bowel Syndrome Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Irritable Bowel Syndrome Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Ulcer Sialic Acid Sialidase Sialic acid Ulcer Sialic Acid Alpha 2,8 Sialidase Sialic acid Ulcer Sialic Acid Alpha 2,3 Sialidase Sialic acid Ulcer Sialic Acid Alpha 2,6 Sialidase Sialic acid Ulcer GalNAc Hexosaminidase GalNAc Ulcer GalNAc Sialidase Hexosaminidase GalNAc Ulcer Sialic acid Hexosaminidase Sialidase Sialic acid Ulcer Galactose galactosidase Galactose Ulcer Galactose sialidase galactosidase Galactose Ulcer Fucose fucosidase Fucose Ulcer Galactose Galactosidase Galactose Ulcer GlcNAc hexosaminidase GlcNAc Ulcer Sulfate Sulfatase Sulfate Ulcer Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Ulcer Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Irritable Bowel Disease Sialic Acid Sialidase Sialic acid Irritable Bowel Disease Sialic Acid Alpha 2,8 Sialidase Sialic acid Irritable Bowel Disease Sialic Acid Alpha 2,3 Sialidase Sialic acid Irritable Bowel Disease Sialic Acid Alpha 2,6 Sialidase Sialic acid Irritable Bowel Disease GalNAc Hexosaminidase GalNAc Irritable Bowel Disease GalNAc Sialidase Hexosaminidase GalNAc Irritable Bowel Disease Sialic acid Hexosaminidase Sialidase Sialic acid Irritable Bowel Disease Galactose galactosidase Galactose Irritable Bowel Disease Galactose sialidase galactosidase Galactose Irritable Bowel Disease Fucose fucosidase Fucose Irritable Bowel Disease Galactose Galactosidase Galactose Irritable Bowel Disease GlcNAc hexosaminidase GlcNAc Irritable Bowel Disease Sulfate Sulfatase Sulfate Irritable Bowel Disease Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Irritable Bowel Disease Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Coronary Artery Disease Sialic Acid Sialidase Sialic acid Coronary Artery Disease Sialic Acid Alpha 2,8 Sialidase Sialic acid Coronary Artery Disease Sialic Acid Alpha 2,3 Sialidase Sialic acid Coronary Artery Disease Sialic Acid Alpha 2,6 Sialidase Sialic acid Coronary Artery Disease GalNAc Hexosaminidase GalNAc Coronary Artery Disease GalNAc Sialidase Hexosaminidase GalNAc Coronary Artery Disease Sialic acid Hexosaminidase Sialidase Sialic acid Coronary Artery Disease Galactose galactosidase Galactose Coronary Artery Disease Galactose sialidase galactosidase Galactose Coronary Artery Disease Fucose fucosidase Fucose Coronary Artery Disease Galactose Galactosidase Galactose Coronary Artery Disease GlcNAc hexosaminidase GlcNAc Coronary Artery Disease Sulfate Sulfatase Sulfate Coronary Artery Disease Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Coronary Artery Disease Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Restenosis Sialic Acid Sialidase Sialic acid Restenosis Sialic Acid Alpha 2,8 Sialidase Sialic acid Restenosis Sialic Acid Alpha 2,3 Sialidase Sialic acid Restenosis Sialic Acid Alpha 2,6 Sialidase Sialic acid Restenosis GalNAc Hexosaminidase GalNAc Restenosis GalNAc Sialidase Hexosaminidase GalNAc Restenosis Sialic acid Hexosaminidase Sialidase Sialic acid Restenosis Galactose galactosidase Galactose Restenosis Galactose sialidase galactosidase Galactose Restenosis Fucose fucosidase Fucose Restenosis Galactose Galactosidase Galactose Restenosis GlcNAc hexosaminidase GlcNAc Restenosis Sulfate Sulfatase Sulfate Restenosis Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Restenosis Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Stroke Sialic Acid Sialidase Sialic acid Stroke Sialic Acid Alpha 2,8 Sialidase Sialic acid Stroke Sialic Acid Alpha 2,3 Sialidase Sialic acid Stroke Sialic Acid Alpha 2,6 Sialidase Sialic acid Stroke GalNAc Hexosaminidase GalNAc Stroke GalNAc Sialidase Hexosaminidase GalNAc Stroke Sialic acid Hexosaminidase Sialidase Sialic acid Stroke Galactose galactosidase Galactose Stroke Galactose sialidase galactosidase Galactose Stroke Fucose fucosidase Fucose Stroke Galactose Galactosidase Galactose Stroke GlcNAc hexosaminidase GlcNAc Stroke Sulfate Sulfatase Sulfate Stroke Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Stroke Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Diabetes Sialic Acid Sialidase Sialic acid Diabetes Sialic Acid Alpha 2,8 Sialidase Sialic acid Diabetes Sialic Acid Alpha 2,3 Sialidase Sialic acid Diabetes Sialic Acid Alpha 2,6 Sialidase Sialic acid Diabetes GalNAc Hexosaminidase GalNAc Diabetes GalNAc Sialidase Hexosaminidase GalNAc Diabetes Sialic acid Hexosaminidase Sialidase Sialic acid Diabetes Galactose galactosidase Galactose Diabetes Galactose sialidase galactosidase Galactose Diabetes Fucose fucosidase Fucose Diabetes Galactose Galactosidase Galactose Diabetes GlcNAc hexosaminidase GlcNAc Diabetes Sulfate Sulfatase Sulfate Diabetes Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Diabetes Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Hyperheparanemia Sialic Acid Sialidase Sialic acid Hyperheparanemia Sialic Acid Alpha 2,8 Sialidase Sialic acid Hyperheparanemia Sialic Acid Alpha 2,3 Sialidase Sialic acid Hyperheparanemia Sialic Acid Alpha 2,6 Sialidase Sialic acid Hyperheparanemia GalNAc Hexosaminidase GalNAc Hyperheparanemia GalNAc Sialidase Hexosaminidase GalNAc Hyperheparanemia Sialic acid Hexosaminidase Sialidase Sialic acid Hyperheparanemia Galactose galactosidase Galactose Hyperheparanemia Galactose sialidase galactosidase Galactose Hyperheparanemia Fucose fucosidase Fucose Hyperheparanemia Galactose Galactosidase Galactose Hyperheparanemia GlcNAc hexosaminidase GlcNAc Hyperheparanemia Sulfate Sulfatase Sulfate Hyperheparanemia Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Hyperheparanemia Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Hypergangliosidemia Sialic Acid Sialidase Sialic acid Hypergangliosidemia Sialic Acid Alpha 2,8 Sialidase Sialic acid Hypergangliosidemia Sialic Acid Alpha 2,3 Sialidase Sialic acid Hypergangliosidemia Sialic Acid Alpha 2,6 Sialidase Sialic acid Hypergangliosidemia GalNAc Hexosaminidase GalNAc Hypergangliosidemia GalNAc Sialidase Hexosaminidase GalNAc Hypergangliosidemia Sialic acid Hexosaminidase Sialidase Sialic acid Hypergangliosidemia Galactose galactosidase Galactose Hypergangliosidemia Galactose sialidase galactosidase Galactose Hypergangliosidemia Fucose fucosidase Fucose Hypergangliosidemia Galactose Galactosidase Galactose Hypergangliosidemia GlcNAc hexosaminidase GlcNAc Hypergangliosidemia Sulfate Sulfatase Sulfate Hypergangliosidemia Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Hypergangliosidemia Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Hypermucinemia Sialic Acid Sialidase Sialic acid Hypermucinemia Sialic Acid Alpha 2,8 Sialidase Sialic acid Hypermucinemia Sialic Acid Alpha 2,3 Sialidase Sialic acid Hypermucinemia Sialic Acid Alpha 2,6 Sialidase Sialic acid Hypermucinemia GalNAc Hexosaminidase GalNAc Hypermucinemia GalNAc Sialidase Hexosaminidase GalNAc Hypermucinemia Sialic acid Hexosaminidase Sialidase Sialic acid Hypermucinemia Galactose galactosidase Galactose Hypermucinemia Galactose sialidase galactosidase Galactose Hypermucinemia Fucose fucosidase Fucose Hypermucinemia Galactose Galactosidase Galactose Hypermucinemia GlcNAc hexosaminidase GlcNAc Hypermucinemia Sulfate Sulfatase Sulfate Hypermucinemia Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Hypermucinemia Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Hyper O-linked Sialic Acid Sialidase Sialic acid glycanemia Hyper O-linked Sialic Acid Alpha 2,8 Sialidase Sialic acid glycanemia Hyper O-linked Sialic Acid Alpha 2,3 Sialidase Sialic acid glycanemia Hyper O-linked Sialic Acid Alpha 2,6 Sialidase Sialic acid glycanemia Hyper O-linked GalNAc Hexosaminidase GalNAc glycanemia Hyper O-linked GalNAc Sialidase Hexosaminidase GalNAc glycanemia Hyper O-linked Sialic acid Hexosaminidase Sialidase Sialic acid glycanemia Hyper O-linked Galactose galactosidase Galactose glycanemia Hyper O-linked Galactose sialidase galactosidase Galactose glycanemia Hyper O-linked Fucose fucosidase Fucose glycanemia Hyper O-linked Galactose Galactosidase Galactose glycanemia Hyper O-linked GlcNAc hexosaminidase GlcNAc glycanemia Hyper O-linked Sulfate Sulfatase Sulfate glycanemia Hyper O-linked Sulfated hexose Sulfatase hexosaminidase GlcNAc or glycanemia GalNAc Hyper O-linked Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA glycanemia acid glucouronidase Hyper N-linked glycanemia Sialic Acid Sialidase Sialic acid Hyper N-linked glycanemia Sialic Acid Alpha 2,8 Sialidase Sialic acid Hyper N-linked glycanemia Sialic Acid Alpha 2,3 Sialidase Sialic acid Hyper N-linked glycanemia Sialic Acid Alpha 2,6 Sialidase Sialic acid Hyper N-linked glycanemia GalNAc Hexosaminidase GalNAc Hyper N-linked glycanemia GalNAc Sialidase Hexosaminidase GalNAc Hyper N-linked glycanemia Sialic acid Hexosaminidase Sialidase Sialic acid Hyper N-linked glycanemia Galactose galactosidase Galactose Hyper N-linked glycanemia Galactose sialidase galactosidase Galactose Hyper N-linked glycanemia Fucose fucosidase Fucose Hyper N-linked glycanemia Galactose Galactosidase Galactose Hyper N-linked glycanemia GlcNAc hexosaminidase GlcNAc Hyper N-linked glycanemia Sulfate Sulfatase Sulfate Hyper N-linked glycanemia Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Hyper N-linked glycanemia Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Hypersialylemia Sialic Acid Sialidase Sialic acid Hypersialylemia Sialic Acid Alpha 2,8 Sialidase Sialic acid Hypersialylemia Sialic Acid Alpha 2,3 Sialidase Sialic acid Hypersialylemia Sialic Acid Alpha 2,6 Sialidase Sialic acid Hypersialylemia GalNAc Hexosaminidase GalNAc Hypersialylemia GalNAc Sialidase Hexosaminidase GalNAc Hypersialylemia Sialic acid Hexosaminidase Sialidase Sialic acid Hypersialylemia Galactose galactosidase Galactose Hypersialylemia Galactose sialidase galactosidase Galactose Hypersialylemia Fucose fucosidase Fucose Hypersialylemia Galactose Galactosidase Galactose Hypersialylemia GlcNAc hexosaminidase GlcNAc Hypersialylemia Sulfate Sulfatase Sulfate Hypersialylemia Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Hypersialylemia Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Hyperfucosylemia Sialic Acid Sialidase Sialic acid Hyperfucosylemia Sialic Acid Alpha 2,8 Sialidase Sialic acid Hyperfucosylemia Sialic Acid Alpha 2,3 Sialidase Sialic acid Hyperfucosylemia Sialic Acid Alpha 2,6 Sialidase Sialic acid Hyperfucosylemia GalNAc Hexosaminidase GalNAc Hyperfucosylemia GalNAc Sialidase Hexosaminidase GalNAc Hyperfucosylemia Sialic acid Hexosaminidase Sialidase Sialic acid Hyperfucosylemia Galactose galactosidase Galactose Hyperfucosylemia Galactose sialidase galactosidase Galactose Hyperfucosylemia Fucose fucosidase Fucose Hyperfucosylemia Galactose Galactosidase Galactose Hyperfucosylemia GlcNAc hexosaminidase GlcNAc Hyperfucosylemia Sulfate Sulfatase Sulfate Hyperfucosylemia Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Hyperfucosylemia Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase Hypersulfogycanemia Sialic Acid Sialidase Sialic acid Hypersulfogycanemia Sialic Acid Alpha 2,8 Sialidase Sialic acid Hypersulfogycanemia Sialic Acid Alpha 2,3 Sialidase Sialic acid Hypersulfogycanemia Sialic Acid Alpha 2,6 Sialidase Sialic acid Hypersulfogycanemia GalNAc Hexosaminidase GalNAc Hypersulfogycanemia GalNAc Sialidase Hexosaminidase GalNAc Hypersulfogycanemia Sialic acid Hexosaminidase Sialidase Sialic acid Hypersulfogycanemia Galactose galactosidase Galactose Hypersulfogycanemia Galactose sialidase galactosidase Galactose Hypersulfogycanemia Fucose fucosidase Fucose Hypersulfogycanemia Galactose Galactosidase Galactose Hypersulfogycanemia GlcNAc hexosaminidase GlcNAc Hypersulfogycanemia Sulfate Sulfatase Sulfate Hypersulfogycanemia Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Hypersulfogycanemia Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase

Provided herein are methods of diagnosing individuals (including, e.g., a disease state or the severity of a disease states) with an infectious disease state associated with abnormal glycan accumulation. Provided in Table 4 are specific embodiments of disease that are optionally diagnosed and/or monitored according to various embodiments described herein. Table 4 also illustrates various non-limiting embodiments of specific enzyme(s) that are optionally utilized to treat a biological sample from an individual suffering from or suspected of (e.g., through a pre- or preliminary screening process) suffering from various infectious disease states associated with abnormal glycan accumulation. Moreover, Table 4 further illustrates various glycan residual compounds that are liberated in various embodiments described herein, such liberated glycan residual compounds optionally being detected and/or measured in order to diagnose and/or monitor various infectious disease states.

TABLE 4 Infectious Diseases Primary Secondary Glycan Non-Reducing Liberating Liberating Residual Disease end structure Enzyme Enzyme Compound Bacterial Infections Mannose Mannosidase Mannose Bacterial Infections Fucose Fucosidase Fucose Bacterial Infections Glucose Glucosidase Glucose Bacterial Infections Galactose Galactosidase Galactose Bacterial Infections GlcNAc hexosaminidase GlcNAc Bacterial Infections GalNAc hexosaminidase GalNAc Bacterial Infections Arabinose Arabinosidase Arabinose Bacterial Infections Xylose Xylosidase Xylose Bacterial Infections Ribose Ribosidase Ribose Bacterial Infections Lyxose Lyxosidase Lyxose Bacterial Infections Talose Talosidase Talose Bacterial Infections Idose Idosidase Idose Bacterial Infections Gulose Gulosidase Gulose Bacterial Infections Altrose Altrosidase Altrose Bacterial Infections Allose Allosidase Allose Fungal Infections Mannose Mannosidase Mannose Fungal Infections Fucose Fucosidase Fucose Fungal Infections Glucose Glucosidase Glucose Fungal Infections Galactose Galactosidase Galactose Fungal Infections GlcNAc hexosaminidase GlcNAc Fungal Infections GalNAc hexosaminidase GalNAc Fungal Infections Arabinose Arabinosidase Arabinose Fungal Infections Xylose Xylosidase Xylose Fungal Infections Ribose Ribosidase Ribose Fungal Infections Lyxose Lyxosidase Lyxose Fungal Infections Talose Talosidase Talose Fungal Infections Idose Idosidase Idose Fungal Infections Gulose Gulosidase Gulose Fungal Infections Altrose Altrosidase Altrose Fungal Infections Allose Allosidase Allose Viral Infections Sialic Acid Sialidase Sialic acid Viral Infections Sialic Acid Alpha 2,8 Sialic acid Sialidase Viral Infections Sialic Acid Alpha 2,3 Sialic acid Sialidase Viral Infections Sialic Acid Alpha 2,6 Sialic acid Sialidase Viral Infections GalNAc Hexosaminidase GalNAc Viral Infections GalNAc Sialidase Hexosaminidase GalNAc Viral Infections Sialic acid Hexosaminidase Sialidase Sialic acid Viral Infections Galactose galactosidase Galactose Viral Infections Galactose sialidase galactosidase Galactose Viral Infections Fucose fucosidase Fucose Viral Infections Galactose Galactosidase Galactose Viral Infections GlcNAc hexosaminidase GlcNAc Viral Infections Sulfate Sulfatase Sulfate Viral Infections Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAc Viral Infections Sulfated uronic Sulfatase Iduronidase or IdoA or GlcA acid glucouronidase

FIG. 1 illustrates compounds present in a normal biological sample not subject to an enzymatic glycan residual liberation process described herein. FIG. 2 illustrates compounds present in a normal biological subject to an enzymatic glycan residual liberation process described herein. FIG. 3 illustrates compounds present in a biological sample of an individual suffering from a disorder associated with abnormal glycan accumulation not subject to an enzymatic glycan residual liberation process described herein. FIG. 4 illustrates compounds present in a biological sample of an individual suffering from a disorder associated with abnormal glycan accumulation subject to an enzymatic glycan residual liberation process described herein.

Detecting and Measuring:

Glycan residual compounds (including, e.g., oligosaccharides, monosaccharides, sulfate, phosphate, sialic acid, acetate, or the like) described herein are detected and/or measured in processes described herein in any suitable manner. In some embodiments, glycan residual compounds are detected and/or measured in unmodified form. In other embodiments, glycan residual compounds are tagged with a detectable label prior and the labeled glycan residual compound is detected.

In some embodiments, non-labeled compounds are optionally detected and/or measured in any suitable manner, e.g., by pH, by quantitative nuclear magnetic resonance (NMR), or the like.

In various embodiments, a method described herein comprises determining whether the amount of liberated glycan residue is abnormal and such a determination comprises labeling the glycan residue with a detectable label and measuring the amount of labeled glycan residue with an analytical instrument. In specific embodiments, the detectable label is a mass label, a radioisotope label, a fluorescent label, a chromophore label, or affinity label. In some embodiments, the amount of liberated glycan is measured using UV-Vis spectroscopy, IR spectroscopy, mass spectrometry, or a combination thereof.

In the various embodiments of any process or method described herein, any suitable detectable label is optionally utilized. In some embodiments, detectable labels useful in the processes or methods described herein include, by way of non-limiting example, mass labels, antibodies, affinity labels, radioisotope labels, chromophores, fluorescent labels, or the like.

Fluorescent labels suitable for use in various embodiments herein include, by way of non-limiting example, 2-aminopyridine (2-AP), 2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), 2-aminoacridone (AMAC), p-aminobenzoic acid ethyl ester (ABEE), p-aminobenzonitrile (ABN), 2-amino-6-cyanoethylpyridine (ACP), 7-amino-4-methylcoumarine (AMC), 8-aminonaphthalene-1,3,6-trisulfate (ANTS),7-aminonaphthalene-1,3-disulfide (ANDS), and 8-aminopyrene-1,3,6-trisulfate (APTS), or the like. The fluorescent labels can be attached by reductive amination with the fluorescent label and sodium cyanoborohydride or the like.

Mass labels suitable for use in various embodiments herein include, by way of non-limiting example, D-2-anthranilic acid, D-2-aminopyridine, D-methyl iodide, ¹³C methyl iodide, deuterated-pyridyl-amine, D-biotin or the like. The mass labels can be attached by permethylation or reductive amination by any method that is known to those of skill in the art.

Affinity labels suitable for use in various embodiments herein include, by way of non-limiting example, biotin and derivatives.

Radioisotope labels suitable for use in various embodiments herein include, by way of non-limiting example, sodium borotritide (NaB³H₄), ³H, ¹⁴C, ³²P, ³⁵S, or the like.

Chromophores suitable for use in various embodiments herein include, by way of non-limiting example, 4-amino-1,1′-azobenzene, 4′-N,N-dimethylamino-4-aminoazobenzene, aminoazobenzene, diaminoazobenzene, Direct Red 16, CI Acid Red 57, CI Acid Blue 45, CI Acid Blue 22, CL Mordant Brown 13, CI Direct Orange 75, or the like. The chromophores may be labeled by any method that is known to those of skill in the art, such as reductive amination with the chromophore and sodium cyanoborohydride.

In some embodiments, the detectable label is an antibody. In specific embodiments, the antibody is attached to a detectable compound, such as mass labels, radioisotope labels, chromophores, fluorescent labels, or the like. In some embodiments, antibodies are themselves detected and/or are detectable in various manners, e.g., as a chromophore, a fluorophore, or the like; or with a probe (e.g., using dot blot techniques, immune-detection techniques, or the like).

In certain embodiments, detectable labels are detected and/or quantified according to any process described herein using any technique, particularly any technique suitable for the detectable label utilized. In some embodiments, suitable detection techniques include, by way of non-limiting example, one or more of a mass spectrometer, a nuclear magnetic resonance spectrometer, a UV-Vis spectrometer, an IR spectrometer, a fluorimeter, a phosphorimeter, a radiation spectrometer (e.g., a scintillation counter), a thin layer chromatographic technique, or the like. In certain embodiments, in any process described herein, glycan residual compounds are optionally directly detected using a suitable technique, such as quantitative nuclear magnetic resonance. Quantitative nuclear magnetic resonance is also optionally utilized to quantify and/or detect the presence of a detectable label. In certain embodiments, one or more glycan residual compounds are optionally detected using a suitable liquid chromatography mass spectrometer (LC-MS).

In some embodiments, glycan residual compounds are tagged with an antibody or probe, and are quantified using any suitable method (e.g., dot blot techniques, immune detection techniques (e.g., ELISA), or the like).

Various analytical methods useful for the processes described herein include, by way of non-limiting example, mass spectrometry, chromatography, HPLC, HPLC, TLC, GC, HPAEC-PAD, electrophoresis capillary or gel, or the like. In certain embodiments, wherein a chromatographic technique is utilized, any suitable solvent system is optionally employed. In certain embodiments, a column (e.g., Cosmogel DEAE, Tsk Gel DEAE, Cosmogel QA, Cosmogel CM, Cosmogel SP, or the like) is optionally loaded with an equilibrating solvent (e.g., a buffer or salt solution, such as a potassium acetate solution, sodium chloride solution, sodium acetate solution, ammonium acetate solution, or the like), e.g., with a pH of about 6, 7, or 8. In some embodiments, the buffer or salt solution has a concentration of about 10 mM, 20 mM, 30 mM, 50 mM, 100 mM, 500 mM, 1 M, 2 M, or the like. Any suitable flow rate is used, e.g., 0.5 mL/min, 1 mL, min, 1.5 mL/min, 2 mL/min, or the like. Following equilibration, a linear gradient is optionally utilized. In some embodiments, the linear gradient is run over 1-20 min, 1-10 min, 10-20 min, 1-5 min, 5-10 min, or the like. In certain embodiments, the gradient is a buffer or salt solution, e.g., as described above (e.g., from 0 M to 0.5 M, from 0 M to 3 M, from 0.5 M to 2 M, from 0 M to 2 M, from 1 M to 2 M, from 0 M to 3 M, from 2 M to 0 M, from 3 M to 0 M, or the like). Once the gradient has reached a final concentration, the eluent is optionally held at the final concentration for a suitable period of time (e.g., 1-20 min, 5-10 min, 10-15 min, 1-5 min, 1-10 min, 15-20 min, or the like). After the optional holding of the final concentration, the eluent may be switched to a second solvent or solvent system (e.g., an alcohol, such as methanol, ethanol, or isopropanol, acetonitrile, water, or the like). The switch to the second solvent system may be over a period of time, e.g., 15 seconds, 30 seconds, 45 seconds, 60 seconds, 2 min, 3 min, or the like. The second solvent system is optionally held for a period of time, such as 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, or the like. Following the second solvent system cycle, the column is optionally restored to initial solvent conditions.

Purification:

In certain embodiments, methods described herein comprise purifying a biological sample, e.g., to remove non-glycan compounds from the biological sample. In some embodiments, a biological sample is purified prior to transforming a glycan thereof.

In certain embodiments, a biological sample containing glycans (purified or not) can also be prepared so that all free glycan residual compounds (e.g., monosaccharides) that are naturally present in the biological sample (i.e., as taken from an individual and without being treated) are eliminated from the sample to reduce background signal (for example using dialysis, spin column, gel filtration, etc).

In some embodiments, any process described herein includes a step of purifying a biological sample comprising removing monosaccharides therefrom, removing sulfates therefrom, removing phosphates therefrom, removing acetate therefrom, removing sialic acid therefrom, or a combination thereof. For example, in some embodiments, a biological sample is optionally placed in to a defined MW cut off spin column (retains large molecules when spun), optionally washed (e.g., with 1 or more volumes of water or buffer), and/or the like.

In certain embodiments, purification of biological samples may further or alternatively comprise, e.g., fractionation, purification, enrichment, or the like of glycans contained therein. In some instances, such purification techniques are suitable to isolate and/or separate different glycan classes within the biological sample prior to transformation of one or more of such glycans. In more specific instances, such purification techniques are used to isolate and/or separate different subsets of a single glycan class (such as isolating complex N-linked glycans from hybrid N-linked structures) prior to transformation of one or more of such glycans. In certain embodiments, a biological sample is optionally prepared in such a way to enrich for specific glycan classes. For example, a PHA affinity column is optionally used to isolate a sub-fraction of complex N-linked glycans while a Con A column could be used to enrich in a different subset of N-linked glycans.

In some embodiments, any process described herein comprises purification of a glycan residual compound resulting from a process described herein (e.g., purification of the glycan residual compound prior to analysis thereof). For example, in some embodiments, the glycan residual compound is optionally isolated by any suitable process, such as by washing the free glycan residual compound (e.g., through a defined MW cut off membrane or by any other suitable method). Moreover, in certain embodiments, the resulting isolated glycan residual compound containing composition is optionally dried or otherwise treated to concentrate the sample and subsequently analyzed for glycan residual compound content by any suitable analytical technique.

In some embodiments, the processes described herein comprises further treatment steps of the test and/or control samples. For example, in some embodiments, the samples are homogenized and/or purified. In specific embodiments homogenization is achieved in any suitable manner including, by way of non-limiting example, with a basic solution, sonication, tissue grinding, or other chemical agents. In some embodiments, severity of a disorder is determined if a certain threshold amount is measured (e.g., as compared to a control or controls) or a threshold signal (e.g., on a fluorimeter or other analytical device utilized to detect and/or measure the generated biomarker). Similarly, a carrier of a disorder described herein is, in certain embodiments, determined if a certain threshold amount is measured (e.g., as compared to a control or controls) or a threshold signal (e.g., on a fluorimeter or other analytical device utilized to detect and/or measure the generated biomarker).

In certain embodiments, samples, including test samples and/or control samples, described herein are optionally purified prior to glycan processing (e.g., lyase treatment) and/or characterization. Test samples and/or control samples (i.e., one or more or all of the glycans found therein) are optionally purified using any suitable purification technique. Test samples and/or control samples are optionally purified at any suitable point in a process described herein, including before or after tagging of the glycans founds within the sample. In certain embodiments, purification techniques include centrifugation, electrophoresis, chromatography (e.g., silica gel or alumina column chromatography), gas chromatography, high performance liquid chromatography (HPLC) (e.g., reverse phase HPLC on chiral or achiral columns), thin layer chromatography, ion exchange chromatography, gel chromatography (e.g., gel filtration or permeation or size exclusion chromatography, gel electrophoresis), molecular sieve chromatography, affinity chromatography, size exclusion, filtration (e.g. through a florisil or activated charcoal plug), precipitation, osmosis, recrystallization, fluorous phase purification, distillation, extraction, chromatofocusing, supercritical fluid extraction, preparative flash chromatography (e.g., flash chromatography using a UV-Vis detector and/or a mass spectrometer (e.g., using the Biotage® suite of products) or the like.

In some embodiments, glycans, such as heparan sulfate, are naturally found attached to a core protein (together forming a proteoglycan) or a lipid. In some embodiments, provided herein are purification processes of separating glycan fragments (e.g., heparan sulfate fragments) from proteoglycans or glycolipids prior to processing the glycan for processing and analysis.

Monitoring Therapy

Provided in certain embodiments are methods of treating disorders associated with the abnormal degradation, biosynthesis and/or accumulation of glycans, the methods comprising:

-   -   a. administering an agent for treating disorders associated with         the abnormal degradation, biosynthesis and/or accumulation of         glycans (e.g., an anti-LSD agent, an anti-cancer agent, or the         like) to an individual in need thereof;     -   b. monitoring the accumulation of glycans in the individual         using any process described herein for detecting or quantifying         the amount of glycan residual compounds (e.g., mono-saccharides,         sulfate, or the like) present in a lyase digested biological         sample (e.g., urine, serum, plasma, or CSF sample) according to         any process described herein.

Provided in further or alternative embodiments are methods of monitoring the treatment of disorders associated with the abnormal degradation, biosynthesis and/or accumulation of glycans, the methods comprising the following steps:

-   -   a. following administration of an agent for treating a disorder         associated with the abnormal degradation, biosynthesis and/or         accumulation of glycans (e.g., an anti-LSD agent, an anti-cancer         agent, or the like) to an individual in need thereof, generating         a biomarker comprising of one or more non-reducing end glycan         residual compound (e.g., monosaccharide).

In some embodiments, the biomarker is a saturated monosaccharide and is generated by treating a population of glycans, in or isolated from a biological sample from the individual, with at least one digesting glycan enzymes, wherein prior to enzyme treatment, the biomarker is not present in abundance in samples from individuals with the disease or condition relative to individuals without the disease or condition. In certain embodiments, monitoring of the accumulation of glycans comprises using an analytical instrument to detect the presence of and/or measure the amount of the biomarker produced and displaying or recording the presence of or a measure of a population of the biomarker; wherein the presence of and/or measure the amount of the biomarker is utilized to monitor the treatment.

In some embodiments, the agent is administered one or more times. In certain embodiments, the agent is administered multiple times. In some embodiments, the agent is administered in a loading dose one or more times (e.g., in a loading dosing schedule) and subsequently administered in a maintenance dose (e.g., in a maintenance dosing schedule, such as three times a day, twice a day, once a day, once every two days, once every three days, once every four days, once a week, or the like). In some embodiments, when glycan (as measure by one or more glycan residual compound(s)) accumulation begins to increase or accelerate, the dose is optionally adjusted (e.g., the maintenance dose is increased, or an additional loading dose or dosing schedule is utilized).

In some embodiments, monitoring the accumulation of glycans comprises repeating the step of: using an analytical instrument to detect the presence of and/or measure the amount of a population of one or more glycan residual compounds present in a transformed biological sample that has been prepared by treating a population of glycans, in or isolated from a biological sample from the individual, with at least one digesting glycan lyase to transform the glycan into the population of the one or more glycan residual compounds. In specific embodiments, the step is repeated at periodic intervals (e.g., every day, every other day, every 2 days, every 3 days, every 4 days, every week, every month, every 3 months, quarterly, every 6 months, yearly, or the like), at regular times following a dose (e.g., 4 hours after a administration of the agent, 6 hours after administration of the agent, 8 hours after administration of the agent, 12 hours after administration of the agent, or the like), prior to administration of the dose (e.g., immediately prior to administration of the agent, 2 hours prior to administration of the agent, or the like), or any other monitoring schedule.

In some embodiments, the monitoring of the accumulation of glycan is conducted over a period of time, e.g., over a week, two weeks, a month, two months, three months, six months, a year, or the like. In some embodiments, the method for quantifying the amount of one or more glycan residual compounds in a lyase digested biological sample (e.g., urine, serum, plasma, or CSF) comprises detecting and/or measuring (e.g., with an analytical device), one or more glycan residual compounds within the lyase digested biological sample from the individual after the biological sample obtained from the individual has been treated with one or more glycan lyases. In certain embodiments, such glycan lyases are suitable for preparing glycan residual compounds from the glycan present in the biological sample obtained from the individual. In certain instances a representative portion of the one or more glycan residual compounds in the transformed biological sample is tagged with any suitable detectable label (e.g., a mass label, a radioisotope label, a fluorescent label, a chromophore label, affinity label, an antibody). In some embodiments, the process comprises displaying or recording such a characterization of the population of glycan residual compounds and/or tagged glycan residual compounds.

In some embodiments, the agent described in a therapy herein includes glycan accumulation inhibitors, agents that promote glycan degradation, agents that activate enzymes that degrade glycans, agents that inhibit biosynthesis of glycans, or the like. In some embodiments, the agent that modulates glycan biosynthesis is an agent that selectively modulates heparan sulfate biosynthesis, an agent that selectively modulates chondroitin sulfate biosynthesis, an agent that selectively modulates dermatan sulfate biosynthesis, an agent that selectively modulates keratan sulfate biosynthesis, an agent that selectively modulates hyaluronan biosynthesis, or a combination thereof. Anti-LSD drugs include, by way of non-limiting example, Imiglucerase (Cerazyme), laronidase (Aldurazyme), idursulfase (Elaprase), galsulfase (Naglazyme), agalsidase beta (Fabrazyme), alglucosidase alfa (Myozyme), agalsidase alfa (Replagal), miglustat (Zavesca).

In some embodiments, one or more of the anti-cancer agents are proapoptotic agents. Examples of anti-cancer agents include, by way of non-limiting example: gossyphol, genasense, polyphenol E, Chlorofusin, all trans-retinoic acid (ATRA), bryostatin, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), 5-aza-2′-deoxycytidine, all trans retinoic acid, doxorubicin, vincristine, etoposide, gemcitabine, imatinib (Gleevec®), geldanamycin, 17-N-Allylamino-17-Demethoxygeldanamycin (17-AAG), flavopiridol, LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, or PD184352, Taxol™, also referred to as “paclitaxel”, which is a well-known anti-cancer drug which acts by enhancing and stabilizing microtubule formation, and analogs of Taxol™, such as Taxotere™. Compounds that have the basic taxane skeleton as a common structure feature, have also been shown to have the ability to arrest cells in the G2-M phases due to stabilized microtubules and may be useful for treating cancer in combination with the compounds described herein.

Further examples of anti-cancer agents include inhibitors of mitogen-activated protein kinase signaling, e.g., U0126, PD98059, PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY 43-9006, wortmannin, or LY294002; Syk inhibitors; mTOR inhibitors; and antibodies (e.g., rituxan).

Other anti-cancer agents include Adriamycin, Dactinomycin, Bleomycin, Vinblastine, Cisplatin, acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; iimofosine; interleukin I1 (including recombinant interleukin II, or r1L2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1; interferon alfa-n3; interferon beta-1a; interferon gamma-1b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazoie; nogalamycin; ormaplatin; oxisuran; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride.

Other anti-cancer agents include: 20-epi-1, 25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; 9-dioxamycin; diphenyl spiromustine; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylerie conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen-binding protein; sizofuran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.

Yet other anticancer agents that include alkylating agents, antimetabolites, natural products, or hormones, e.g., nitrogen mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, etc.), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne, ete.), or triazenes (decarbazine, etc.). Examples of antimetabolites include but are not limited to folic acid analog (e.g., methotrexate), or pyrimidine analogs (e.g., Cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin).

Examples of natural products include but are not limited to vinca alkaloids (e.g., vinblastin, vincristine), epipodophyllotoxins (e.g., etoposide), antibiotics (e.g., daunorubicin, doxorubicin, bleomycin), enzymes (e.g., L-asparaginase), or biological response modifiers (e.g., interferon alpha).

Examples of alkylating agents include, but are not limited to, nitrogen mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, meiphalan, etc.), ethylenimine and methylmelamines (e.g., hexamethlymelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne, semustine, streptozocin, etc.), or triazenes (decarbazine, ete.). Examples of antimetabolites include, but are not limited to folic acid analog (e.g., methotrexate), or pyrimidine analogs (e.g., fluorouracil, floxouridine, Cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin.

Examples of hormones and antagonists include, but are not limited to, adrenocorticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate), estrogens (e.g., diethlystilbestrol, ethinyl estradiol), antiestrogen (e.g., tamoxifen), androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogen (e.g., flutamide), gonadotropin releasing hormone analog (e.g., leuprolide). Other agents that can be used in the methods and compositions described herein for the treatment or prevention of cancer include platinum coordination complexes (e.g., cisplatin, carboblatin), anthracenedione (e.g., mitoxantrone), substituted urea (e.g., hydroxyurea), methyl hydrazine derivative (e.g., procarbazine), adrenocortical suppressant (e.g., mitotane, aminoglutethimide).

In some instances, the detection and/or the quantification of the identity and/or amount of glycan residual compounds present in a biological sample is used to identify and/or diagnose a disorder associated with abnormal degradation, biosynthesis and/or accumulation of glycan in an individual suspected of having such a disorder.

In some instances, the detection and/or the quantification of the identity and/or amount of glycan residual compounds present in the biological sample is used to monitor severity and course of the disease in an individual diagnosed with or suspected of having a disorder associated with the abnormal degradation, biosynthesis and/or accumulation of glycans. In some instances, the detection and/or the quantification of the identity and/or amount of glycan residual compounds present in the biological sample is used to calculate the administered dose of an agent that modulates (e.g., promotes and/or inhibits) glycan biosynthesis and/or degradation.

In certain instances, wherein following administration of a selected dose of a therapeutic agent utilized in a therapeutic method described herein, an individual's condition does not improve, the detection and/or the quantification of the identity and/or amount of glycan residual compounds present in a biological sample provides for a treatment regimen to be modified depending on the severity and course of the disease, disorder or condition, previous therapy, the individual's health status and response to the drugs, and the judgment of the treating physician.

In certain embodiments, monitoring the accumulation of glycans in the individual comprises detecting or quantifying the amount of an glycan residual compounds (or one or more glycan residual compounds) in a sample obtained from the individual (e.g., according to any method described herein) to obtain a first accumulation result (e.g., an initial reading before treatment has begun, or at any other time) and a second accumulation result that is subsequent to obtaining the first result. In some embodiments, the second result is compared to the first result to determine if the treatment is effectively reducing, maintaining, or reducing the rate of increasing the glycan residual compounds levels in a substantially identically obtained sample from the individual being treated. In certain embodiments, depending on the difference between the first and second results, the treatment can be altered, e.g., to increase or decrease the amount of agent administered; to substitute the therapeutic agent with an alternative therapeutic agent; or the like. In certain embodiments, the dose of the therapeutic agent is decreased to a maintenance level (e.g., if the glycan residual compound level has been reduced sufficiently); further monitoring of glycan residual compound levels is optional in such situation, e.g., to ensure that reduced or maintained levels of glycan residual compounds (e.g., monosaccharide(s)) are achieved.

Alternatively, provided herein is a method of detecting response to therapy in an individual or a method of predicting response to therapy in an individual comprising:

-   -   a. administering an agent for treating a disorder associated         with the abnormal degradation, biosynthesis and/or accumulation         of glycans to a plurality of cells from an individual in need         thereof (e.g., a plurality of fibroblasts, serum, plasma, or CSF         cells from a human suffering from a disorder associated with the         abnormal degradation, biosynthesis and/or accumulation of         glycans, such as an LSD or cancer);     -   b. monitoring the accumulation of glycans in the plurality of         cells using any process described herein for detecting or         quantifying the amount of glycan residual compounds (e.g.,         monosaccharides, sulfate, sialic acid, phosphate, acetate, or         the like) present in a lyase digested biological sample from the         plurality of cells according to any process described herein.

In specific embodiments, the glycan residual compound(s) detected or measured is one or more monosaccharide. It is to be understood that a plurality of cells from an individual includes cells that are directly taken from the individual, and/or cells that are taken from an individual followed by culturing to expand the population thereof.

EXAMPLES Example 1

To illustrate the methods described herein, we have used human urine sample from normal patients and patients diagnosed with MPS IIIA. MPS IIIA patients have reduced function of the lysosomal enzyme that de-N-sulfates the nonreducing end glucosamine residues present in heparan sulfate. This unique nonreducing end glycan residual (N-sulfated GlcN) can be liberated by treating the glycans with heparin lyases and quantified by fluorescent detection on HPLC. As shown below, glycans prepared in this manner from normal individuals lack N-sulfate G1cN while MPS IIIA patients have a very high level.

Purification: The biological sample (cells, tissue, blood, serum, or the like) is homogenized and solublized in 0.1-1.0 N NaOH (e.g., 0.1 N, 0.2 N, 0.3 N, 0.4 N, 0.5 N, 0.6 N, 0.7 N, 0.8 N, 0.9 N, or 1.0 N) or acetic acid and then neutralized with acetic acid or NaOH. Next a small sample is taken to measure protein content of the sample using standard methods. 0.01-0.5 mg/mL (0.01 mg/mL, 0.07 mg/mL, 0.12 mg/mL, 0.17 mg/mL, 0.22 mg/mL, 0.27 mg/mL, 0.32 mg/mL, 0.37 mg/mL, 0.42 mg/mL, or 0.5 mg/mL) protease (trypsin, chymotrypsin, pepsin, pronase, papain, or elastase) is treated in 0.1-0.5 M (e.g., 0.1 M, 0.16 M, 0.23 M, 0.32 M, 0.39 M, 0.44 M, or 0.5 M) NaCl, 0.01-0.1 M (e.g., 0.01 M, 0.02 M, 0.04 M, 0.06 M, 0.08 M, 0.1 M) NaOAc, at pH 5.5-7.5 (e.g., 5.5, 6.0, 6.5, 7.0, or 7.5) and 25-40 C (e.g., 25 C, 30 C, 35 C, or 40 C) for 1-24 hours (e.g., 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 18 h, 24 h). The sample is diluted to reduce the ionic strength and loaded onto an ion exchange column in 5-100 mM (e.g., 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 75 mM, 80 mM, 90 mM, 95 mM, 100 mM) NaOAc pH 5-7 with 0-300 mM NaCl. After washing, the bound glycosaminoglycans are eluted with 5-100 mM NaOAc pH 5-7 (e.g., 5, 5.5, 6, 6.5, 7) with 0.8-3 M (e.g., 0.8 M, 1 M, 1.2 M, 1.4 M, 1.6 M, 1.8 M, 2 M, 2.5 M, or 3 M) NaCl. The eluted glycans are then concentrated and desalted by ethanol precipitation, size exclusion, or other methods. The purified glycans are dried for further analysis.

Liberation of non-reducing end residual: The purified glycans are resuspended in 10-300 mM sodium acetate, tris, phosphate, or other suitable buffer, 0.02-1 mM (e.g., 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) calcium acetate, pH 5-8 (e.g., 5, 5.5, 6, 6.5, 7, 7.5, or 8), were digested with heparin lyases I, II, III, I and II, I and III, II and III, or I, II, and III (0.0.15-1.5 milliunits of each in 100-ul reactions, IBEX, Montreal, Canada) at 25 to 37° C. for 1 to 24 hours.

Fluorescent tagging of glycan residual: Dried glycan sample is re-suspended in 2-100 μL 0.003-0.1 M (e.g., 0.003 M, 0.003 M, 0.03 M, 0.06 M, 0.1 M) AB, AA, AMAC, or Bodipy dye and incubated at room temperature for 1-120 minutes (e.g., 1-10 min, 10-15 min, 15-20 min, 20-25 min, 25-30 min, 30-40 min, 40-50 min, 50-60 min, 60-90 min, 90-120 min). Next, the reaction is initiated with 2-100 μL (2 μL, 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, or 100 μL) 1 M NaCNBH₄ and the reaction is allowed to proceed at 25-100 C. (e.g., 25 C, 30 C, 35 C, 40 C, 50 C, 60 C, 70 C, 80 C, 90 C, 100 C).

Detection of glycan residual: HPLC separation of tagged saccharides was performed utilizing the following conditions: Column types: 130A BEH particle Phenyl (1.7, 2.5, 3.5, 5, or 10 uM particle size), 130A BEH particle C18 (1.7, 2.5, 3.5, 5, or 10 uM particle size), HSS particle C18 (1.8, 3.5, or 5 uM particle size), or 300A BEH particle C18 (1.7, 3.5, 5, 10 uM particle size) with suitable length and internal diameter.

Buffer Conditions:

-   -   A=Ammonium Acetate, Sodium Acetate, or Sodium Chloride (e.g., 0         M, 10 mM, 20 mM, 30 mM, 40 mM, 100 mM, 500 mM, 1 M, 2 M) with         0-20% methanol     -   B=100% Alcohol, such as methanol, ethanol, or isopropanol     -   Initial Conditions: 70-95% A, 0-30% B     -   Flow Rate is constant at 0.05-1 ml/min     -   Runs a gradient down to 70-90% A, 10-30% B over 5-65 min.     -   At 8.1 min runs a gradient to 0-20% A, 80-100% B over 5-20 min.     -   5-65 min returns to initial conditions

FIG. 1 illustrates an HPLC trace of eluted compounds detected in normal patient urine not subject to enzymatic glycan residual liberation (i.e., providing background signals). FIG. 2 illustrates an HPLC trace of eluted compounds detected in normal patient urine subject to enzymatic glycan residual liberation as set forth in Example 1. FIG. 3 illustrates an HPLC trace of eluted compounds detected in MPS IIIA patient urine not subject to enzymatic glycan residual liberation (i.e., providing background signals). FIG. 4 illustrates an HPLC trace of eluted compounds detected in MPS IIIA patient urine subject to enzymatic glycan residual liberation.

Example 2

The processes described in Example 1 are repeated and/or modified for the diseases listed in Tables 1-4 utilizing the enzymes described there in and detecting the glycan residual compounds also described therein. 

1. A method of diagnosing an individual as having a disease or condition associated with abnormal glycan biosynthesis, degradation, or accumulation, the method comprising: (a) generating a biomarker comprising of one or more non-reducing end glycan residual compound, wherein the biomarker is generated by treating a population of glycans, in or isolated from a biological sample from the individual, with at least one digesting glycan enzymes, wherein prior to enzyme treatment, the biomarker is not present in abundance in samples from individuals with the disease or condition relative to individuals without the disease or condition, and (b) using an analytical instrument to detect the presence of and/or measure the amount of the biomarker produced and displaying or recording the presence of or a measure of a population of the biomarker; wherein the presence of and/or measure the amount of the biomarker is utilized to determine the presence, identity, and/or severity of the disease or condition.
 2. The method of claim 1, wherein the disease or disorder is caused by an abnormally functioning glycan degradation enzyme and wherein the abnormally functioning glycan degradation enzyme and the normally functioning glycan degradation enzyme are of the same type.
 3. The method of claim 1, wherein the abnormally functioning glycan degradation enzyme functions abnormally as a result of being present in an abnormally low amount, functioning improperly, or a combination thereof.
 4. The method of claim 1, wherein the abnormal glycan accumulation comprises the accumulation of abnormal amounts of glycans.
 5. The method of claim 1, wherein the abnormal glycan accumulation comprises the accumulation of abnormal amounts of normal glycans.
 6. The method of claim 1, wherein the abnormal glycan accumulation comprises the accumulation of abnormal amounts of abnormal glycans.
 7. The method of claim 1, wherein the normally functioning glycan degradation enzyme is a glycosidase, sulfatase, phosphorylase, deacetylase, or a combination thereof.
 8. The method of claim 7, wherein the normally functioning glycan degradation enzyme is a glycosidase selected from an exo-glycosidase and an endo-glycosidase.
 9. The method of claim 8 wherein the glycosidase is an exo-glycosidase selected from the group consisting of a galactosidase, and a glucuronidase.
 10. The method of claim 8, wherein the glycan residual compound is a monosaccharide.
 11. The method of claim 7, wherein the glycan residual compound is sulfate, phosphate, acetate, or a combination thereof.
 12. The method of claim 1, further comprising purifying a biological sample prior to transforming a glycan thereof.
 13. The method of claim 12, wherein the process of purifying a biological sample comprises removing monosaccharides therefrom, removing sulfates therefrom, removing phosphates therefrom, removing acetate therefrom, or a combination thereof.
 14. The method of claim 1, wherein transforming a glycan of a biological sample with a normally functioning glycan degradation enzyme comprises transforming a glycan of a biological sample with a plurality of normally functioning glycan degradation enzymes.
 15. The method of claim 14, wherein the glycan is treated with a plurality of normally functioning glycan degradation enzymes concurrently, sequentially, or a combination thereof.
 16. The method of claim 1, wherein the disorder associated with an abnormal glycan accumulation is MPS I, MPS II, MPS IIIA, MPS IVA, MPSVI, or Fabry Disease.
 17. The method of claim 1, wherein determining whether the amount of liberated glycan residue is abnormal comprises labeling the glycan residue with a detectable label and measuring the amount of labeled glycan residue with an analytical instrument.
 18. The method of claim 17, wherein the detectable label is a mass label, a radioisotope label, a fluorescent label, a chromophore label, or affinity label.
 19. The method of claim 17, wherein the amount of liberated glycan is measured using UV-Vis spectroscopy, IR spectroscopy, mass spectrometry, or a combination thereof.
 20. A method monitoring the treatment of a disorder associated with the abnormal degradation, biosynthesis and/or accumulation of glycans, the method comprising: (a) following administration of an agent for treating a disorder associated with the abnormal degradation, biosynthesis and/or accumulation of glycans to an individual in need thereof, using an analytical instrument to measure the amount of a population of a biomarker comprising a non-reducing end glycan residual compounds present in a transformed biological sample, the biomarker being generated by treating a population of glycans, in or isolated from a biological sample from the individual, with at least one digesting glycan enzyme(s), wherein prior to enzyme treatment, the biomarker is not present in abundance in samples from individuals with the disease or condition relative to individuals without the disease or condition, and (b) determining whether or not the amount of the amount of biomarker has decreased or increased at a slower rate compared to the amount or rate of increase prior to administration of the agent for treating a disorder associated with the abnormal degradation, biosynthesis and/or accumulation of glycans.
 21. The method of claim 20, wherein the disorder associated with the abnormal degradation, biosynthesis and/or accumulation of glycans is a lysosomal storage disease, a cancerous disease, an inflammatory disease, an infectious disease, a central nervous system disease, or a cardiovascular disease.
 22. The method of claim 20, wherein the normally functioning glycan degradation enzyme is a glycosidase, sulfatase, phosphorylase, deacetylase, or a combination thereof.
 23. The method of claim 22, wherein the normally functioning glycan degradation enzyme is a glycosidaseis selected from an exo-glycosidase and an endo-glycosidase.
 24. The method of claim 20, wherein the glycan residual compound is a monosaccharide, sulfate, phosphate, acetate, or a combination thereof.
 25. The method of claim 20, wherein transforming a glycan of a biological sample with a normally functioning glycan degradation enzyme comprises transforming a glycan of a biological sample with a plurality of normally functioning glycan degradation enzymes.
 26. The method of claim 25, wherein the glycan is treated with a plurality of normally functioning glycan degradation enzymes concurrently, sequentially, or a combination thereof.
 27. The method of claim 1, wherein prior to measuring the amount of a population of non-reducing end glycan residual compounds, the non-reducing end glycan residual compounds are labeled with a detectable label.
 28. The method of claim 27, wherein the detectable label is a mass label, a radioisotope label, a fluorescent label, a chromophore label, or affinity label.
 29. The method of claim 27, wherein the amount of liberated glycan is measured using UV-Vis spectroscopy, IR spectroscopy, mass spectrometry, or a combination thereof. 